Method of cloning animals

ABSTRACT

The present invention relates to cloning technologies. The invention relates in part to immortalized and totipotent cells useful for cloning animals, the embryos produced from these cells using nuclear transfer techniques, animals that arise from these cells and embryos, and materials, methods, and processes for establishing such cells, embryos, and animals.

This application is a continuation of Ser. No. 09/354,276 filed Jul. 15,1999 now U.S. Pat. No. 6,395,958 and is a continuation of Ser. No.09/239,922 filed Jan. 28, 1999 now U.S. Pat. No. 6,011,197 and is acontinuation-in-part of U.S. Application Serial No. 60/073,019, filedJan. 29, 1998, entitled “Cloning of Biological Organisms fromImmortalized Totipotent Cells” (pending); U.S. application Ser. No.08/812,851, filed Mar. 6, 1997, entitled “Method of Cloning Animals”(pending); P.C.T Application Serial No. PCT/US 98/04345, filed Mar. 5,1998, entitled “Method of Cloning Animals” (pending); and U.S.application Ser. No. 08/812,031, filed Mar. 6, 1997, entitled “Method ofCloning Bovines” (pending), each of which is hereby incorporated byreference in its entirety including any drawings, and from each of whichpriority is claimed.

FIELD OF THE INVENTION

The invention relates to the cloning of animals.

BACKGROUND OF THE INVENTION

The following discussion of the background of the invention is merelyprovided to aid the reader in understanding the invention and is notadmitted to describe or constitute prior art to the present invention.

Researchers have been developing methods for cloning mammalian animalsover the past two decades. These reported methods typically include thesteps of (1) isolating a cell, most often an embryonic cell; (2)inserting the cell or nucleus isolated from the cell into an enucleatedoocyte (e.g., the oocyte's nucleus was previously extracted), and (3)allowing the embryo to mature in vivo.

The first successful nuclear transfer experiment using mammalian cellswas reported in 1983, where the pronuclei isolated from a murine (mouse)zygote were inserted into an enucleated oocyte and resulted in likeoffspring(s). McGrath & Solter, 1983, Science 220:1300-1302.Subsequently, others described the production of chimeric murine embryos(e.g., embryos that contain a subset of cells having significantlydifferent nuclear DNA from other cells in the embryo) using murineprimordial germ cells (PGC). These cells are and can give rise topluripotent cells (e.g., cells that can differentiate into other typesof cells but do not differentiate into a grown animal). Matsui et al.,1992, Cell 70:841-847 and Resnick et al., 1992, Nature 359:550; Kato etal., 1994, Journal of Reproduction and Fertility Abstract Series,Society For the Study of Fertility, Annual Conference, Southampton,13:38.

Some publications related to murine pluripotent cells stress theimportance of steel factor for converting precursor cells intopluripotent cells. U.S. Pat. Nos. 5,453,357 and 5,670,372, entitled“Pluripotent Embryonic Stem Cells and Methods of Making Same,” issued toHogan. These same publications indicate that murine pluripotent cellsexhibit strong, uniform alkaline phosphatase staining.

Although murine animals were never clearly cloned from nuclear transfertechniques using embryonic cells, some progress was reported in thefield of cloning ovine (sheep) animals. One of the first successfulnuclear transfer experiments utilizing ovine embryonic cells as nucleardonors was reported in 1986. Willadsen, 1986, Nature 320:63-65. A decadelater, others reported that additional lambs were cloned from ovineembryonic cells. Campbell et al., 1996, Nature 380:64-66 and PCTPublication WO 95/20042. Recently, another lamb was reported to becloned from ovine somatic mammary tissue. Wilmut et al., 1997, Nature385:810-813. Some methods for cloning ovine animals focused uponutilizing serum deprived somatic ovine cells and cells isolated fromovine embryonic discs as nuclear donors. PCT Publications WO 96/07732and WO 97/07669. Other methods for cloning ovine animals involvedmanipulating the activation state of an in vivo matured oocyte afternuclear transfer. PCT Publication WO 97/07668.

While few lambs were produced, publications that disclose cloned lambsreport a cloning efficiency that is, at best, approximately 0.4%.Cloning efficiency, as calculated for the previous estimate, is a ratioequal to the number of cloned lambs divided by the number of nucleartransfers used to produce that number of cloned lambs.

Despite the slower progress endemic to the field of cloning bovineanimals, a bovine animal was cloned using embryonic cells derived from2-64 cell embryos. This bovine animal was cloned by utilizing thenuclear transfer techniques set forth in U.S. Pat. Nos. 4,994,384 and5,057,420. Others reported that cloned bovine embryos were formed bynuclear transfer techniques utilizing the inner cell mass cells of ablastocyst stage embryo. Sims & First, 1993, Theriogenology 39:313 andKeefer et al., 1994, Mol. Reprod. Dev. 38:264-268. In addition, anotherpublication reported that cloned bovine embryos were prepared by nucleartransfer techniques that utilized PGCs isolated from fetal tissue.Delhaise et al., 1995, Reprod. Fert. Develop. 7:1217-1219; Lavoir 1994,J. Reprod. Dev. 37:413-424; and PCT application WO 95/10599 entitled“Embryonic Stem Cell-Like Cells.” However, the reports demonstrated thatcloned PGC-derived bovine embryos never clearly developed past the firsttrimester during gestation. Similarly, embryonic stem cell (e.g., cellline derived from embryos which are undifferentiated, pluripotent, andcan establish a permanent cell line which exhibits a stable karyotype),ESC, derived bovine embryos never developed past fifty-five days,presumably due to incomplete placental development. Stice et al., 1996,Biol. Reprod. 54: 100-110.

Despite the progress of cloning ovine and bovine animals, there remainsa great need in the art for methods and materials that increase cloningefficiency. In addition there remains a great need in the art to expandthe variety of cells that can be utilized as nuclear donors, especiallyexpanding nuclear donors to non-embryonic cells. Furthermore, thereremains a long felt need in the art for karyotypically stable permanentcell lines that can be used for genome manipulation and production oftransgenic cloned animals.

SUMMARY

The present invention relates to cloning technologies. The inventionrelates in part to immortalized, totipotent cells useful for cloninganimals, the embryos produced from these cells using nuclear transfertechniques, animals that arise from these cells and embryos, and themethods and processes for creating such cells, embryos, and animals.

The present invention provides multiple advantages over the tools andmethods currently utilized in the field of mammalian cloning. Suchfeatures and advantages include:

(1) Production of cloned animals from virtually any type of cell. Theinvention provides materials and methods for reprogrammingnon-totipotent cells into totipotent cells. These non-totipotent cellsmay be of non-embryonic origin. This feature of the invention allows forthe ability to assess the phenotype of an existing animal and thenreadily establish a permanent cell line for cloning that animal.

(2) Creation of permanent cell lines from virtually any type of cell.Permanent cell lines provide a nearly unlimited source of geneticmaterial for nuclear transfer cloning techniques. In one aspect of theinvention, non-totipotent precursor cells can be reprogrammed intototipotent and permanent cells. These non-totipotent precursor cells maybe non-embryonic cells. Permanent cell lines provide the advantage ofenhancing cloning efficiency due to the lower cellular heterogeneitywithin the cell lines (e.g., permanent cells that have lower rates ofdifferentiation than primary culture cell lines currently used forcloning). In addition, the permanent cell lines can be manipulated invitro to produce cells, embryos, and animals whose genomes have beenmanipulated (e.g., transgenic). Furthermore, permanent cell lines can bemore easily stored, transported, and re-established in culture thanother types of cell lines.

(3) Enhancement of the efficiency for cloning embryos as a result ofutilizing asynchronous, permanent, and karyotypically stable cell linesin a complete in vitro embryo production system.

Cloning efficiency can be expressed by the ratio between the number ofembryos resulting from nuclear transfer and the number of nucleartransfers performed to give rise to the embryos. Alternatively, cloningefficiency can be expressed as the ratio between the number of live bornanimals and the number of nuclear transfers performed to give rise tothese animals.

Immortalized and Totipotent Cells of the Invention

In a first aspect, the invention features a totipotent mammalian cell.Preferably, the totipotent mammalian cell is (1) a cultured cell; (2) acell cultured in a cell line; and (3) an immortalized cell. In addition,the mammalian cell is preferably an ungulate cell and more preferably abovine cell.

The term “mammalian” or “mammal” as used herein refers to any animal ofthe class Mammalia. A mammalian animal of the invention is preferably anendangered animal, or, more preferably, a farm animal. Most preferably,a mammal is an ungulate.

The term “non-ovine” as used herein refers to any animal other than ananimal of the family Ovidae. Members of the family Oviadae includesheep. A non-ovine mammal is any member of the class Mammalia other thanan animal of the family Ovidae. Preferable non-ovine animals areungulate animals and most preferably are bovine and porcine animals.

The term “ungulate” as used herein refers to a four-legged animal havinghooves. In other preferred embodiments, the ungulate is selected fromthe group consisting of domestic or wild representatives of bovids,ovids, cervids, suids, equids and camelids. Examples of suchrepresentatives are cows or bulls, bison, buffalo, sheep, big-hornsheep, horses, ponies, donkeys, mule, deer, elk, caribou, goat, waterbuffalo, camels, llama, alpaca, and pigs. Especially preferred in thebovine species are Bos taurus, Bos indicus, and Bos buffaloes cows orbulls.

The term “bovine” as used herein refers to a family of ruminantsbelonging to the genus Bos or any closely related genera of the familyBovidae. The family Bovidae includes true antelopes, oxen, sheep, andgoats, for example. Preferred bovine animals are the cow and ox.Especially preferred bovine species are Bos taurus, Bos indicus, and Bosbuffaloes. Other preferred bovine species are Bos primigenius and Boslongifrons.

The term “totipotent” as used herein refers to a cell that gives rise toall of the cells in a developing cell mass, such as an embryo, fetus,and animal. In preferres embodiments, the term “totipotent” also refersto a cell that gives rise to all of the cells in an animal. A totipotentcell can give rise to all of the cells of a developing cell mass when itis utilized in a procedure for creating an embryo from one or morenuclear transfer steps. An animal may be an animal that functions exutero. An animal can exist, for example, as a live born animal.Totipotent cells may also be used to generate incomplete animals such asthose useful for organ harvesting, e.g., having genetic modifications toeliminate growth of a head such as by manipulation of a homeotic gene.

The terms “developing cell mass” as used herein refers to a group ofcells in which all cells or a portion of the cells are undergoing celldivision. The developing cell mass may be an embryo, a fetus, and/or ananimal, for example. The developing cell mass may be dividing in vitro(e.g., in culture) or in vivo (e.g., in utero). The developing cell massmay be a product of one or more nuclear transfer processes or may be theproduct of oocyte activation (e.g., sperm mediated fertilization).

The term “live born” as used herein preferably refers to an animal thatexists ex utero. A “live born” animal may be an animal that is alive forat least one second from the time it exits the maternal host. A “liveborn” animal may not require the circulatory system of an in uteroenvironment for survival. A “live born” animal may be an ambulatoryanimal. Such animals can include pre- and post-pubescent animals. Inaddition, a “live born animal” may also be deceased for a certain periodof time. As discussed previously, a “live born” animal may lack aportion of what exists in a normal animal of its kind. For example, a“live born” animal may lack a head as a result of the deletion ormanipulation of one or more homeotic genes.

The term “totipotent” as used herein is to be distinguished from theterm “pluripotent.” The latter term refers to a cell that differentiatesinto a sub-population of cells within a developing cell mass, but is acell that may not give rise to all of the cells in that developing cellmass. Thus, the term “pluripotent” can refer to a cell that cannot giverise to all of the cells in a live born animal.

The term “totipotent” as used herein is also to be distinguished fromthe term “chimer” or “chimera.” The latter term refers to a developingcell mass that comprises a sub-group of cells harboring nuclear DNA witha significantly different nucleotide base sequence than the nuclear DNAof other cells in that cell mass. The developing cell mass can, forexample, exist as an embryo, fetus, and/or animal.

The term “immortalized” or “permanent” as used herein in reference tocells refers to cells that have exceeded the Hayflick limit. TheHayflick limit can be defined as the number of cell divisions that occurbefore a cell line becomes senescent. Hayflick set this limit toapproximately 60 divisions for most non-immortalized cells. See, e.g.,Hayflick and Moorhead, 1961, Exp. Cell. Res. 25: 585-621; and Hayflick,1965, Exp. Cell Research 37: 614-636, incorporated herein by referencein their entireties including all figures, tables, and drawings.Therefore, an immortalized cell line can be distinguished fromnon-immortalized cell lines if the cells in the cell line are able toundergo more than 60 divisions. If the cells of a cell line are able toundergo more than 60 cell divisions, the cell line is an immortalized orpermanent cell line. The immortalized cells of the invention arepreferably able to undergo more than 70 divisions, are more preferablyable to undergo more than 80 divisions, and are most preferably able toundergo more than 90 cell divisions.

Typically, immortalized or permanent cells can be distinguished fromnon-immortalized and non-permanent cells on the basis that immortalizedand permanent cells can be passaged at densities lower than those ofnon-immortalized cells. Specifically, immortalized cells can be grown toconfluence (e.g., when a cell monolayer spreads across an entire plate)when plating conditions do not allow physical contact between the cells.Hence, immortalized cells can be distinguished from non-immortalizedcells when cells are plated at cell densities where the cells do notphysically contact one another.

The term “plated” or “plating” as used herein in reference to cellsrefers to establishing cell cultures in vitro. For example, cells can bediluted in cell culture media and then added to a cell culture plate orcell culture dish. Cell culture plates are commonly known to a person ofordinary skill in the art. Cells may be plated at a variety ofconcentrations and/or cell densities.

The meaning of the term “cell plating” can also extend to the term “cellpassaging.” Immortalized cells of the invention can be passaged usingcell culture techniques well known to those skilled in the art. The term“cell passaging” can refer to such techniques which typically involvethe steps of (1) releasing cells from a solid support and disassociationof these cells, and (2) diluting the cells in fresh media suitable forcell proliferation. Immortalized cells can be successfully grown byplating the cells in conditions where they lack cell to cell contact.Cell passaging may also refer to removing a portion of liquid mediumbathing cultured cells and adding liquid medium from another source tothe cell culture.

The term “proliferation” as used herein in reference to immortalized orpermanent cells refers to a group of cells that can increase in sizeand/or can increase in numbers over a period of time.

The term “confluence” as used herein refers to a group of cells where alarge percentage of the cells are physically contacted with at least oneother cell in that group. Confluence may also be defined as a group ofcells that grow to a maximum cell density in the conditions provided.For example, if a group of cells can proliferate in a monolayer and theyare placed in a culture vessel in a suitable growth medium, they areconfluent when the monolayer has spread across a significant surfacearea of the culture vessel. The surface area covered by the cellspreferably represents about 50% of the total surface area, morepreferably represents about 70% of the total surface area, and mostpreferably represents about 90% of the total surface area.

The term “culture” as used herein in reference to cells refers to one ormore cells that are undergoing cell division or not undergoing celldivision in an in vitro environment. An in vitro environment can be anymedium known in the art that is suitable for maintaining cells in vitro,such as suitable liquid media or agar. Specific examples of suitable invitro environments for cell cultures are described in Culture of AnimalCells: a manual of basic techniques (3^(rd) edition), 1994, R. I.Freshney (ed.), Wiley-Liss, Inc.; Cells: a laboratory manual (vol. 1),1998, D. L. Spector, R. D. Goldman, L. A. Leinwand (eds.), Cold SpringHarbor Laboratory Press; and Animal Cells: culture and media, 1994, D.C. Darling, S. J. Morgan, John Wiley and Sons, Ltd., each of which isincorporated herein by reference in its entirety including all figures,tables, and drawings. Preferred media are AminoMax™-C100 Basal Medium(Gibco 1701-082), AminoMax™ C-100 Supplement Medium (Gibco 17002-080),and Knockout™ D-MEM Medium (Gibco 10829-108).

Nearly any type of cell can be placed in cell culture conditions. Cellsmay be cultured in suspension and/or in monolayers with one or moresubstantially similar cells. Cells may be cultured in suspension and/orin monolayers with a heterogeneous population cells. The term“heterogeneous” as utilized in the previous sentence can relate to anycell characteristics, such as cell type and cell cycle stage, forexample. Cells may be cultured in suspension and/or in monolayers withfeeder cells. The term “feeder cells” is defined hereafter. In preferredembodiments, cells may be successfully cultured by plating the cells inconditions where they lack cell to cell contact. Cell cultures can alsobe utilized to establish a cell line.

In preferred embodiments, (1) cultured cells undergo cell division; (2)cells are cultured for greater than 5 hours; (3) cells are cultured forgreater than 7 hours; (4) cells are cultured for greater than 10 hours;(5) cells are cultured for greater than 12 hours; (6) cells are culturedfor greater than 24 hours; (7) cells are cultured for and greater than48 hours; (8) cells are cultured greater than 3 days; (9) cells arecultured for greater than 5 days; (10) cells are cultured for greaterthan 10 days; and (11) cells are cultured for greater than 30 days.

The term “suspension” as used herein refers to cell culture conditionsin which the cells are not attached to a solid support. Cellsproliferating in suspension can be stirred while proliferating usingapparatus well known to those skilled in the art.

The term “monolayer” as used herein refers to cells that are attached toa solid support while proliferating in suitable culture conditions. Asmall portion of the cells proliferating in the monolayer under suitablegrowth conditions may be attached to cells in the monolayer but not tothe solid support. Preferably less than 15% of these cells are notattached to the solid support, more preferably less than 10% of thesecells are not attached to the solid support, and most preferably lessthan 5% of these cells are not attached to the solid support. Cells canalso grow in culture in multilayers. The term “multilayer” as usedherein refers to cells proliferating in suitable culture conditionswhere at least 15% of the cells are indirectly attached to the solidsupport through an attachment to other cells. Preferably, at least 25%of the cells are indirectly attached to the solid support, morepreferably at least 50% of the cells are indirectly attached to thesolid support, and most preferably at least 75% of the cells areindirectly attached to the solid support.

The term “substantially similar” as used herein in reference toimmortalized bovine cells refers to cells from the same organism and thesame tissue. In preferred embodiments, substantially similar also refersto cell populations that have not significantly differentiated. Forexample, preferably less than 15% of the cells in a population of cellshave differentiated, more preferably less than 10% of the cellpopulation have differentiated, and most preferably less than 5% of thecell population have differentiated.

The term “cell line” as used herein refers to cultured cells that can bepassaged more than once. The invention relates to cell lines that can bepassaged more than 2, 5, 6, 7, 8, 9, 10, 15, 20, 30, 50, 80, 100, and200 times, or preferably more than any integer between 2 and 200, eachnumber not having been explicitly set forth in the interest ofconciseness. The concept of cell passaging is defined previously.

In preferred embodiments, (1) the totipotent cells are not alkalinephosphatase positive; (2) the totipotent cells arise from at least oneprecursor cell; (3) the precursor cell is isolated from and/or arisesfrom any region of an animal; (4) the precursor cell is isolated fromand/or arises from any cell in culture; (5) the precursor cell isselected from the group consisting of a non-embryonic cell, a non-fetalcell, a differentiated cell, a somatic cell, an embryonic cell, a fetalcell, an embryonic stem cell, a primordial germ cell, a genital ridgecell, an amniotic cell, a fetal fibroblast cell, an ovarian follicularcell, a cumulus cell, an hepatic cell, an endocrine cell, an endothelialcell, an epidermal cell, an epithelial cell, a fibroblast cell, ahematopoietic cell, a keratinocyte, a renal cell, a lymphocyte, amelanocyte, a muscle cell, a myeloid cell, a neuronal cell, anosetoblast, a mesenchymal cell, a mesodermal cell, an adherent cell, acell isolated from an asynchronous population of cells, and a cellisolated from a synchronized population of -cells where the synchronouspopulation is not arrested in the G₀ stage of the cell cycle; and (6)the precursor cell is preferably isolated and/or arises from a mammaliananimal, more preferably an ungulate animal, and most preferably a bovineanimal.

The term “alkaline phosphatase positive” as used herein refers to adetectable presence of cellular alkaline phosphatase. Cells that are notalkaline phosphatase positive do not stain appreciably using a procedurefor visualizing cellular alkaline phosphatase. Procedures for detectingthe presence of cellular alkaline phosphatase are well-known to a personof ordinary skill in the art. See, e.g., Matsui et al., 1991, “Effect ofSteel Factor and Leukemia Inhibitory Factor on Murine Primordial GermCells in Culture,” Nature 353: 750-752. Examples of cells that stainappreciably for alkaline phosphatase can be found in the art. See, e.g.,U.S. Pat. No. 5,453,357, Entitled “Pluripotent Embryonic Stem Cells andMethods of Making Same,” issued to Hogan on Sep. 26, 1995, which isincorporated by reference herein in its entirety, including all figures,tables, and drawings.

The term “precursor cell” or “precursor cells” as used herein refers toa cell or cells used to create a cell line of totipotent cells. The cellline is preferably permanent. Precursor cells can be isolated from anymammal, preferably from an ungulate and more preferably from a bovineanimal. The precursor cell or cells may be isolated from nearly anycellular entity. For example, a precursor cell or cells may be isolatedfrom blastocysts, embryos, fetuses, and cell lines (e.g., cell linesestablished from embryonic cells), preferably isolated from fetusesand/or cell lines established from fetal cells, and more preferablyisolated from ex utero animals and/or cell cultures and/or cell linesestablished from such ex utero animals. An ex utero animal may exist as,a newborn animal, adolescent animal, yearling animal, and adult animal.The ex utero animals may be alive or post mortem. The precursor cell orcells may be immortalized or non-immortalized. These examples are notmeant to be limiting and a further description of these exemplaryprecursor cells is provided hereafter.

The term “arises from” as used herein refers to the conversion of one ormore cells into one or more other cells. For example, a non-totipotentprecursor cell can be converted into a totipotent cell by utilizingfeatures of the invention described hereafter. This conversion processcan be referred to as a reprogramming step. In another example, aprecursor cell can give rise to a feeder layer of cells, as definedhereafter. In addition, the term “arises from” can refer to the creationof totipotent embryos from immortalized, totipotent cells of theinvention, as described hereafter.

The term “reprogramming” or “reprogrammed” as used herein refers tomaterials and methods that can convert a non-totipotent cell into antotipotent cell. Distinguishing features between totipotent andnon-totipotent cells are described previously. An example of materialsand methods for converting non-totipotent cells into totipotent cells isto incubate precursor cells with a receptor ligand cocktail. Receptorligand cocktails are described hereafter.

The term “isolated” as used herein refers to a cell that is mechanicallyseparated from another group of cells. Examples of a group of cells area developing cell mass, a cell culture, a cell line, and an animal.These examples are not meant to be limiting and the invention relates toany group of cells.

The term “non-embryonic cell” as used herein refers to a cell that isnot isolated from an embryo. Non-embryonic cells can be differentiatedor non-differentiated. Non-embryonic cells can refer to nearly anysomatic cell, such as cells isolated from an ex utero animal. Theseexamples are not meant to be limiting.

For the purposes of the present invention, the term “embryo” or“embryonic” as used herein refers to a developing cell mass that has notimplanted into the uterine membrane of a maternal host. Hence, the term“embryo” as used herein can refer to a fertilized oocyte, a cybrid(defined herein), a pre-blastocyst stage developing cell mass, and/orany other developing cell mass that is at a stage of development priorto implantation into the uterine membrane of a maternal host. Embryos ofthe invention may not display a genital ridge. Hence, an “embryoniccell” is isolated from and/or has arisen from an embryo.

An embryo can represent multiple stages of cell development. Forexample, a one cell embryo can be referred to as a zygote, a solidspherical mass of cells resulting from a cleaved embryo can be referredto as a morula, and an embryo having a blastocoel can be referred to asa blastocyst.

The term “fetus” as used herein refers to a developing cell mass thathas implanted into the uterine membrane of a maternal host. A fetus caninclude such defining features as a genital ridge, for example. Agenital ridge is a feature easily identified by a person of ordinaryskill in the art, and is a recognizable feature in fetuses of mostanimal species. The term “fetal cell” as used herein can refer to anycell isolated from and/or has arisen from a fetus or derived from afetus. The term “non-fetal cell” is a cell that is not derived orisolated from a fetus.

The term “primordial germ cell” as used herein refers to a diploidsomatic cell capable of becoming a germ cell. Primordial germ cells canbe isolated from the genital ridge of a developing cell mass. Thegenital ridge is a section of a developing cell mass that is well-knownto a person of ordinary skill in the art. See, e.g., Strelchenko, 1996,Theriogenology 45: 130-141 and Lavoir 1994, J. Reprod. Dev. 37: 413-424.

The terms “embryonic germ cell” and “EG cell” as used herein refers to acultured cell that has a distinct flattened morphology and can growwithin monolayers in culture. An EG cell may be distinct from afibroblast cell. This EG cell morphology is to be contrasted with cellsthat have a spherical morphology and form multicellular clumps on feederlayers. Embryonic germ cells may not require the presence of feederlayers or presence of growth factors in cell culture conditions.Embryonic germ cells may also grow with decreased doubling rates whenthese cells approach confluence on culture plates. Embryonic germ cellsof the invention may be totipotent. Embryonic germ cells of theinvention may not appreciably stain for alkaline phosphatase.Preferably, embryonic germ cells are established in culture media thatcontains a significant concentration of glucose.

Embryonic germ cells may be established from a cell culture of nearlyany type of precursor cell. Examples of precursor cells are discussedherein, and a preferred precursor cell for establishing an embryonicgerm cell culture is a genital ridge cell from a fetus. Genital ridgecells are preferably isolated from porcine fetuses where the fetus isbetween 20 days and parturition, between 30 days and 100 days, morepreferably between 35 days and 70 days and between 40 days and 60 days,and most preferably about a 55 day fetus. An age of a fetus can bedetermined as described above. The term “about” with respect to fetusescan refer to plus or minus five days. As described herein, EG cells maybe physically isolated from a primary culture of cells, and theseisolated EG cells may be utilized to establish a cell culture thateventually forms a homogenous or nearly homogenous line of EG cells.

The term “embryonic stem cell” as used herein refers to pluripotentcells isolated from an embryo that are maintained in in vitro cellculture. Embryonic stem cells may be cultured with or without feedercells. Embryonic stem cells can be established from embryonic cellsisolated from embryos at any stage of development, including blastocyststage embryos and pre-blastocyst stage embryos. Embryonic stem cells arewell known to a person of ordinary skill in the art. See, e.g., WO97/37009, entitled “Cultured Inner Cell Mass Cell-Lines Derived fromUngulate Embryos,” Stice and Golueke, published Oct. 9, 1997, and Yang &Anderson, 1992, Theriogenology 38: 315-335, both of which areincorporated herein by reference in their entireties, including allfigures, tables, and drawings.

The term “amniotic cell” as used herein refers to any cultured ornon-cultured cell isolated from amniotic fluid. Examples of methods forisolating and culturing amniotic cells are discussed in Bellow et al.,1996, Theriogenology 45: 225; Garcia & Salaheddine, 1997, Theriogenology47: 1003-1008; Leibo & Rail, 1990, Theriogenology 33: 531-552; and Voset al., 1990, Vet. Rec. 127: 502-504, each of which is incorporatedherein by reference in its entirety, including all figures tables anddrawings. Particularly preferred are cultured amniotic cells that arespherical (e.g., cultured amniotic cells that do not display afibroblast-like morphology). Also preferred amniotic cells are fetalfibroblast cells. The terms “fibroblast,” fibroblast-like,” “fetal,” and“fetal fibroblast” are defined hereafter.

The terms “fibroblast-like” and “fibroblast” as used herein refer tocultured cells that have a distinct flattened morphology and that areable to grow within monolayers in culture.

The term “fetal fibroblast cell” as used herein refers to anydifferentiated fetal cell having a fibroblast appearance. Whilefibroblasts characteristically have a flattened appearance when culturedon culture media plates, fetal fibroblast cells can also have aspindle-like morphology. Fetal fibroblasts may require densitylimitation for growth, may generate type I collagen, and may have afinite life span in culture of approximately fifty generations.Preferably, fetal fibroblast cells rigidly maintain a diploidchromosomal content. For a description of fibroblast cells, see, e.g.,Culture of Animal Cells: a manual of basic techniques (3^(rd) edition),1994, R. I. Freshney (ed), Wiley-Liss, Inc., incorporated herein byreference in its entirety, including all figures, tables, and drawings.

The terms “morphology” and “cell morphology” as used herein refer toform, structure, and physical characteristics of cells. For example, onecell morphology is significant levels of alkaline phosphatase, and thiscell morphology can be identified by determining whether a cell stainsappreciably for alkaline phosphatase. Another example of a cellmorphology is whether a cell is flat or round in appearance whencultured on a surface or in the presence of a layer of feeder cells.Many other cell morphologies are known to a person of ordinary skill inthe art and are cell morphologies are readily identifiable usingmaterials and methods well known to those skilled in the art. See, e.g.,Culture of Animal Cells: a manual of basic techniques (3^(rd) edition),1994, R. I. Freshney (ed.), Wiley-Liss, Inc.

The term “ovarian follicular cell” as used herein refers to a culturedor non-cultured cell obtained from an ovarian follicle, other than anoocyte. Follicular cells may be isolated from ovarian follicles at anystage of development, including primordial follicles, primary follicles,secondary follicles, growing follicles, vesicular follicles, maturingfollicles, mature follicles, and graafian follicles. Furthermore,follicular cells may be isolated when an oocyte in an ovarian follicleis immature (i.e., an oocyte that has not progressed to metaphase II) orwhen an oocyte in an ovarian follicle is mature (i.e., an oocyte thathas progressed to metaphase II or a later stage of development).Preferred follicular cells include, but are not limited to, pregranulosacells, granulosa cells, theca cells, columnar cells, stroma cells, thecainterna cells, theca externa cells, mural granulosa cells, luteal cells,and corona radiata cells. Particularly preferred follicular cells arecumulus cells. Various types of follicular cells are known and can bereadily distinguished by those skilled in the art. See, e.g., LaboratoryProduction of Cattle Embryos, 1994, Ian Gordon, CAB International;Anatomy and Physiology of Farm Animals (5th ed.), 1992, R. D. Frandsonand T. L. Spurgeon, Lea & Febiger, each of which is incorporated hereinby reference in its entirety including all figures, drawings, andtables. Individual types of follicular cells may be cultured separately,or a mixture of types may be cultured together.

The term “cumulus cell” as used herein refers to any cultured ornon-cultured cell isolated from cells and/or tissue surrounding anoocyte. Persons skilled in the art can readily identify cumulus cells.Examples of methods for isolating and/or culturing cumulus cells arediscussed in Damiani et al., 1996, Mol. Reprod. Dev. 45: 521-534; Longet al., 1994, J. Reprod. Fert. 102: 361-369; and Wakayama et al., 1998,Nature 394: 369-373, each of which is incorporated herein by referencein its entireties, including all figures, tables, and drawings. Cumuluscells may be isolated from ovarian follicles at any stage ofdevelopment, including primordial follicles, primary follicles,secondary follicles, growing follicles, vesicular follicles, maturingfollicles, mature follicles, and graafian follicles. Cumulus cells maybe isolated from oocytes in a number of manners well known to a personof ordinary skill in the art. For example, cumulus cells can beseparated from oocytes by pipeting the cumulus cell/oocyte complexthrough a small bore pipette, by exposure to hyaluronidase, or bymechanically disrupting (e.g. vortexing) the cumulus cell/oocytecomplex. Additionally, exposure to Ca⁺⁺/Mg⁺⁺ free media can removecumulus from immature oocytes. Also, cumulus cell cultures can beestablished by placing matured oocytes in cell culture media. Oncecumulus cells are removed from media containing increased LH/FSHconcentrations, they can to attach to the culture plate.

The term “hepatic cell” as used herein refers to any cultured ornon-cultured cell isolated from a liver. Particularly preferred hepaticcells include, but are not limited to, a hepatic parenchymal cell, aKüpffer cell, an Ito cell, a hepatocyte, a fat-storing cell, a pit cell,and a hepatic endothelial cell. Persons skilled in the art can readilyidentify the various types of hepatic cells. See, e.g., Regulation ofHepatic Metabolism, 1986, Thurman et al. (eds.), Plenum Press, which isincorporated herein by reference in its entirety including all figures,drawings, anid tables.

The term “differentiated cell” as used herein refers to a precursor cellthat has developed from an unspecialized phenotype to that of aspecialized phenotype. For example, embryonic cells can differentiateinto an epithelial cell lining the intestine. It is highly unlikely thatdifferentiated cells revert into their precursor cells in vivo or invitro. However, materials and methods of the invention can reprogramdifferentiated cells into immortalized, totipotent cells. Differentiatedcells can be isolated from a fetus or a live born animal, for example.

In contrast to the totipotent and/or immortalized cells of the inventionthat arise from non-embryonic cells, an example of embryonic cells isdiscussed in WO 96/07732, entitled “Totipotent Cells for NuclearTransfer,” hereby incorporated herein by reference in its entiretyincluding all figures, drawings, and tables. The WO 96/07732 publicationrelates primarily to ovine animals. A unique feature of the presentinvention is that immortalized, totipotent cells are reprogrammed fromnon-embryonic cells by utilizing the materials and methods describedherein in descriptions of the preferred embodiments and exemplaryembodiments.

The term “asynchronous population” as used herein refers to cells thatare not arrested at any one stage of the cell cycle. Many cells canprogress through the cell cycle and do not arrest at any one stage,while some cells can become arrested at one stage of the cell cycle fora period of time. Some known stages of the cell cycle are G₀, G₁, S, G₂,and M. An asynchronous population of cells is not manipulated tosynchronize into any one or predominantly into any one of these phases.Cells can be arrested in the G₀ stage of the cell cycle, for example, byutilizing multiple techniques known in the art, such as by serumdeprivation. Examples of methods for arresting non-immortalized cells inone part of the cell cycle are discussed in WO 97/07669, entitled“Quiescent Cell Populations for Nuclear Transfer,” hereby incorporatedherein by reference in its entirety, including all figures, tables, anddrawings.

The terms “synchronous population” and “synchronizing” as used hereinrefer to a fraction of cells in a population that are arrested (i.e.,the cells are not dividing) in a discreet stage of the cell cycle.Synchronizing a population of cells, by techniques such as serumdeprivation, may render the cells quiescent. The term “quiescent” isdefined below. Preferably, about 50% of the cells in a population ofcells are arrested in one stage of the cell cycle, more preferably about70% of the cells in a population of cells are arrested in one stage ofthe cell cycle, and most preferably about 90% of the cells in apopulation of cells are arrested in one stage of the cell cycle. Cellcycle stage can be distinguished by relative cell size as well as by avariety of cell markers well known to a person of ordinary skill in theart. For example, cells can be distinguished by such markers by usingflow cytometry techniques well known to a person of ordinary skill inthe art. Alternatively, cells can be distinguished by size utilizingtechniques well known to a person of ordinary skill in the art, such asby the utilization of a light microscope and a micrometer, for example.

The terms “serum deprivation,” “serum starved,” and “serum starvation”as used herein refer to culturing cells in a medium comprising a serumconcentration sufficiently low as to render cultured cells quiescent.The term “quiescent” is defined hereafter. A number of sera are used bythose skilled in the art to supplement cell culture media. Particularlypreferred is fetal bovine serum. Preferred serum starvation conditionsare culturing cells in a medium comprising less than 1% fetal bovineserum. Particularly preferred conditions are culturing cells in a mediumcomprising not more than 0.5% fetal bovine serum. A length of timecultured cells are serum starved to be rendered quiescent can varydepending upon cell type. Cultured cells can be serum starved for atleast 1 hour, at least 5 hours, at least 12 hours, and at least 24hours. Preferably, cultured cells are serum starved for more than 1 day.Most preferably, cultured cells are serum starved for more than 3 days.These conditions are not meant to be limiting, and other serumstarvation conditions can easily be identified by those skilled in theart without undue experimentation.

The term “quiescent” as used herein in reference to cells refers tocells which are not dividing. A “quiescent cell culture” refers to aculture in which a majority of cells in the culture are not dividing.More preferably, in a quiescent cell culture all cells in the cultureare not dividing. As discussed herein, a cell culture may be renderedquiescent by serum starvation, but other methods which render cellcultures quiescent are known to those of ordinary skill in the art.Cells may be made permanently quiescent, and more preferably, quiescentcells may be made to resume dividing at a later time.

In preferred embodiments, (1) the totipotent cells of the inventioncomprise modified nuclear DNA; (2) the modified nuclear DNA includes aDNA sequence that encodes a recombinant product; (3) the recombinantproduct is a polypeptide; (4) the recombinant product is a ribozyme; (4)the recombinant product is expressed in a biological fluid or tissue;(5) the recombinant product confers or partially confers resistance toone or more diseases; (6) the recombinant product confers resistance orpartially confers resistance to one or more parasites; (7) the modifiednuclear DNA comprises at least one other DNA sequence that can functionas a regulatory element; (8) the regulatory element is selected from thegroup consisting of promotor, enhancer, insulator, and repressor; and(9) the regulatory element is selected from the group consisting of milkprotein promoter, urine protein promoter, blood protein promoter, tearduct protein promoter, synovial protein promoter, mandibular glandprotein promoter, casein promoter, β-casein promoter, melanocortinpromoter, milk serum protein promoter, α-lactalbumin promoter, whey acidprotein promoter, uroplakin promoter, α-actin promoter.

The term “modified nuclear DNA” as used herein refers to the nucleardeoxyribonucleic acid sequence of a cell, embryo, fetus, or animal ofthe invention that has been manipulated by one or more recombinant DNAtechniques. Examples of these recombinant DNA techniques are well knownto a person of ordinary skill in the art, which can include (1)inserting a DNA sequence from another organism (e.g., a human organism)into target nuclear DNA, (2) deleting one or more DNA sequences fromtarget nuclear DNA, and (3) introducing one or more base mutations(e.g., site-directed mutations) into target nuclear DNA. Cells withmodified nuclear DNA can be referred to as “transgenic cells” for thepurposes of the invention. Transgenic cells can be useful as materialsfor nuclear transfer cloning techniques provided herein.

Methods and tools for insertion, deletion, and mutation of nuclear DNAof mammalian cells are well-known to a person of ordinary skill in theart. See, Molecular Cloning, a Laboratory Manual, 2nd Ed., 1989,Sambrook, Fritsch, and Maniatis, Cold Spring Harbor Laboratory Press;U.S. Pat. No. 5,633,067, “Method of Producing a Transgenic Bovine orTransgenic Bovine Embryo,” DeBoer et al., issued May 27, 1997; U.S. Pat.No. 5,612,205, “Homologous Recombination in Mammalian Cells,” Kay etal., issued Mar. 18, 1997; and PCT publication WO 93/22432, “Method forIdentifying Transgenic Pre-Implantation Embryos,” all of which areincorporated by reference herein in their entirety, including allfigures, drawings, and tables. These methods include techniques fortransfecting cells with foreign DNA fragments and the proper design ofthe foreign DNA fragments such that they effect insertion, deletion,and/or mutation of the target DNA genome.

Transgenic cells may be obtained in a variety of manners. For example,transgenic cells can be isolated from a transgenic animal. Examples oftransgenic animals are well known in the art, as described herein withregard to transgenic bovine and ovine animals. Cells isolated from atransgenic animal can be converted into totipotent and/or immortalizedcells by using the materials and methods provided herein. In anotherexample, transgenic cells can be created from totipotent and/orimmortalized cells of the invention. Materials and methods forconverting non-transgenic cells into transgenic cells are well known inthe art, as described previously.

Any of the cell types defined herein can be altered to harbor modifiednuclear DNA. For example, embryonic stem cells, fetal cells, and anytotipotent and immortalized cell defined herein can be altered to harbormodified nuclear DNA.

Examples of methods for modifying a target DNA genome by insertion,deletion, and/or mutation are retroviral insertion, artificialchromosome techniques, gene insertion, random insertion with tissuespecific promoters, homologous recombination, gene targeting,transposable elements, and/or any other method for introducing foreignDNA. Other modification techniques well known to a person of ordinaryskill in the art include deleting DNA sequences from a genome, and/oraltering nuclear DNA sequences. Examples of techniques for alteringnuclear DNA sequences are site-directed mutagenesis and polymerase chainreaction procedures. Therefore, the invention provides for bovine cellsthat are simultaneously totipotent, immortalized, and transgenic. Thesetransgenic, totipotent, immortalized cells can serve as nearly unlimitedsources of donor cells for production of cloned transgenic animals.

The term “recombinant product” as used herein refers to the productproduced from a DNA sequence that comprises at least a portion of themodified nuclear DNA. This product can be a peptide, a polypeptide, aprotein, an enzyme, an antibody, an antibody fragment, a polypeptidethat binds to a regulatory element (a term described hereafter), astructural protein, an RNA molecule, and/or a ribozyme, for example.These products are well defined in the art. This list of products is forillustrative purposes only and the invention relates to other types ofproducts.

The term “ribozyme” as used herein refers to ribonucleic acid moleculesthat can cleave other RNA molecules in specific-regions. Ribozymes canbind to discrete regions on a RNA molecule, and then specifically cleavea region within that binding region or adjacent to the binding region.Ribozyme techniques can thereby decrease the amount of polypeptidetranslated from formerly intact message RNA molecules. For specificdescriptions of ribozymes, see U.S. Pat. No. 5,354,855, entitled “RNARibozyme which Cleaves Substrate RNA without Formation of a CovalentBond,” Cech et al., issued on Oct. 11, 1994, and U.S. Pat. No.5,591,610, entitled “RNA Ribozyme Polymerases, Dephosphorylases,Restriction Endoribonucleases and Methods,” Cech et al., issued on Jan.7, 1997, both of which are incorporated by reference in their entiretiesincluding all figures, tables, and drawings.

The term “biological fluid or tissue” as used herein refers to any fluidor tissue in a biological organism. The fluids may include, but are notlimited to, tears, saliva, milk, urine, amniotic fluid, semen, plasma,oviductal fluid, and synovial fluid. The tissues may include, but arenot limited to, lung, heart, blood, liver, muscle, brain, pancreas,skin, and others.

The term “confers resistance” as used herein refers to the ability of arecombinant product to completely abrogate or partially alleviate thesymptoms of a disease or parasitic condition. Hence, if the disease isrelated to inflammation, for example, a recombinant product can conferresistance to that inflammation if the inflammation decreases uponexpression of the recombinant product. A recombinant product may conferresistance or partially confer resistance to a disease or parasiticcondition, for example, if the recombinant product is an anti-sense RNAmolecule that specifically binds to an mRNA molecule encoding apolypeptide responsible for the inflammation. Other examples ofconferring resistance to diseases or parasites are described hereafter.In addition, examples of diseases are described hereafter.

Examples of parasites and strategies for conferring resistance to theseparasites are described hereafter. These examples include, but are notlimited to, worms, insects, invertebrate, bacterial, viral, andeukaryotic parasites. These parasites can lead to diseased states thatcan be controlled by the materials and methods of the invention.

The term “regulatory element” as used herein refers to a DNA sequencethat can increase or decrease the amount of product produced fromanother DNA sequence. The regulatory element can cause the constitutiveproduction of the product (e.g., the product can be expressedconstantly). Alternatively, the regulatory element can enhance ordiminish the production of a recombinant product in an inducible fashion(e.g., the product can be expressed in response to a specific signal).The regulatory element can be controlled, for example, by nutrition, bylight, or by adding a substance to the transgenic organism's system.Examples of regulatory elements well-known to those of ordinary skill inthe art are promoters, enhancers, insulators, and repressors. See, e.g.,Transgenic Animals, Generation and Use, 1997, Edited by L. M. Houdebine,Hardwood Academic Publishers, Australia, hereby incorporated herein byreference in its entirety including all figures, tables, and drawings.

The term “promoters” or “promoter” as used herein refers to a DNAsequence that is located adjacent to a DNA sequence that encodes arecombinant product. A promoter is preferably operatively linked to theadjacent DNA sequence. A promoter typically increases the amount ofrecombinant product expressed from a DNA sequence as compared to theamount of the expressed recombinant product when no promoter exists. Apromoter from one organism can be utilized to enhance recombinantproduct expression from a DNA sequence that originates from anotherorganism. In addition, one promoter element can increase an amount ofrecombinant products expressed for multiple DNA sequences attached intandem. Hence, one promoter element can enhance the expression of one ormore recombinant products. Multiple promoter elements are well-known topersons of ordinary skill in the art. Examples of promoter elements aredescribed hereafter.

The term “enhancers” or “enhancer” as used herein refers to a DNAsequence that is located adjacent to the DNA sequence that encodes arecombinant product. Enhancer elements are typically located upstream ofa promoter element or can be located downstream of the coding DNAsequence (e.g., the DNA sequence transcribed or translated into arecombinant product or products). Hence, an enhancer element can belocated 100 base pairs, 200 base pairs, or 300 or more base pairsupstream of the DNA sequence that encodes the recombinant product.Enhancer elements can increase the amount of recombinant productexpressed from a DNA sequence above the increased expression afforded bya promoter element. Multiple enhancer elements are readily available topersons of ordinary skill in the art.

The term “insulators” or “insulator” as used herein refers to DNAsequences that flank the DNA sequence encoding the recombinant product.Insulator elements can direct the recombinant product expression tospecific tissues in an organism. Multiple insulator elements are wellknown to persons of ordinary skill in the art. See, e.g., Geyer, 1997,Curr. Opin. Genet. Dev. 7: 242-248, hereby incorporated herein byreference in its entirety, including all figures, tables, and drawings.

The term “repressor” or “repressor element” as used herein refers to aDNA sequence located in proximity to the DNA sequence that encodes therecombinant product, where the repressor sequence can decrease theamount of recombinant product expressed from that DNA sequence.Repressor elements can be controlled by the binding of a specificmolecule or specific molecules to the repressor element DNA sequence.These molecules can either activate or deactivate the repressor element.Multiple repressor elements are available to a person of ordinary skillin the art.

The terms “milk protein promoter,” “urine protein promoter,” “bloodprotein promoter,” “tear duct protein promoter,” “synovial proteinpromoter,” and “mandibular gland protein promoter” refer to promoterelements that regulate the specific expression of proteins within thespecified fluid or gland or cell type in an animal. For example, a milkprotein promoter is a regulatory element that can control the expressionof a protein that is expressed in the milk of an animal. Otherpromoters, such as casein promoter, α-lactalbumin promoter, whey acidprotein promoter, uroplakin promoter, arid α-actin promoter, forexample, are well known to a person of ordinary skill in the art.

In preferred embodiments, (1) the totipotent cell is subject tomanipulation; (2) the manipulation comprises the step of utilizing atotipotent cell in a nuclear transfer procedure; (3) the manipulationcomprises the step of cryopreserving the totipotent cells; (4) themanipulation comprises the step of thawing the totipotent cells; (5) themanipulation comprises the step of passaging totipotent cells; (6) themanipulation comprises the step of synchronizing totipotent cells; (7)the manipulation comprises the step of transfecting totipotent cellswith foreign DNA; and (8) the manipulation comprises the step ofdissociating a cell from another cell or group of cells.

The term “manipulation” as used herein refers to the common usage of theterm, which is the management or handling directed towards some object.Examples of manipulations are described herein.

The term “nuclear transfer” as used herein refers to introducing a fullcomplement of nuclear DNA from one cell to an enucleated cell. Nucleartransfer methods are well known to a person of ordinary skill in theart. See, U.S. Pat. No. 4,994,384, entitled “Multiplying BovineEmbryos,” Prather et al., issued on Feb. 19, 1991 and U.S. Pat. No.5,057,420, entitled “Bovine Nuclear Transplantation,” Massey, issued onOct. 15, 1991, both of which are hereby incorporated by reference intheir entirety including all figures, tables and drawings. Nucleartransfer may be accomplished by using oocytes that are not surrounded bya zona pellucida.

Although the basic principals of nuclear transfer have been describedpreviously, the technique can be sensitive to the introduction of anynew parameters. Therefore, significant modifications to the techniquesdescribed in the area of nuclear transfer may require someexperimentation to determine the practical effect of these modificationsupon the efficiency of nuclear transfer. An example of a variable thatcan affect nuclear transfer efficiency is the age of the oocyte utilizedfor enucleation and nuclear transfer.

The term “cryopreserving” as used herein refers to freezing a cell,embryo, or animal of the invention. The cells, embryos, or portions ofanimals of the invention are frozen at temperatures preferably lowerthan 0° C., more preferably lower than −80° C., and most preferably attemperatures lower than −196° C. Cells and embryos in the invention canbe cryopreserved for an indefinite amount of time. It is known thatbiological materials can be cryopreserved for more than fifty years. Forexample, semen that is cryopreserved for more than fifty years can beutilized to artificially inseminate a female bovine animal. Methods andtools for cryopreservation are well-known to those skilled in the art.See, e.g., U.S. Pat. No. 5,160,312, entitled “Cryopreservation Processfor Direct Transfer of Embryos,” issued to Voelkel on Nov. 3, 1992.

The term “thawing” as used herein refers to the process of increasingthe temperature of a cryopreserved cell, embryo, or portions of animals.Methods of thawing cryopreserved materials such that they are activeafter the thawing process are well-known to those of ordinary skill inthe art.

The terms “transfected,” “transformation,” and “transfection” as usedherein refer to methods of inserting foreign DNA into a cellularorganism. These methods involve a variety of techniques, such astreating the cells with high concentrations of salt, an electric field,liposomes, polycationic micelles, or detergent, to render the host cellouter membrane or wall permeable to nucleic acid molecules of interest.Transfection techniques are well known to a person of ordinary skill inthe art and materials and methods for carrying out transfection of DNAconstructs into cells are commercially available. Materials typicallyused to transfect cells with DNA constructs are lipophilic compoundssuch as Lipofectin™. Particular lipophilic compounds can be induced toform liposomes for mediating transfection of the DNA construct into thecells. These specified methods are not limiting and the inventionrelates to any transformation technique well known to a person ofordinary skill in the art. See, e.g., Molecular Cloning, a LaboratoryManual, 2nd Ed., 1989, Sambrook, Fritsch, and Maniatis, Cold SpringHarbor Laboratory Press and Transgenic Animals, Generation and Use,1997, Edited by L. M. Houdebine, Hardwood Academic Publishers,Australia, both of which were previously incorporated by reference.

The term “foreign DNA” as used herein refers to DNA that can betransfected into a target cell, where the foreign DNA harbors at leastone base pair modification as compared to the nuclear DNA of the targetorganism. Foreign DNA and transfaction can be further understood anddefined in conjunction with the term “modified nuclear DNA,” describedpreviously.

The term “dissociating” as used herein refers to the materials andmethods useful for pulling a cell away from another cell. For example, ablastomere (i.e., a cellular member of a blastocyst stage embryo) can bepulled away from the rest of the developing cell mass by techniques andapparatus well known to a person of ordinary skill in the art. See,e.g., U.S. Pat. No. 4,994,384, entitled “Multiplying Bovine Embryos,”issued on Feb. 19, 1991, hereby incorporated herein by reference in itsentirety, including all figures, tables, and drawings. Alternatively,cells proliferating in culture can be separated from one another tofacilitate such processes as cell passaging, which is describedpreviously. In addition, dissociation of a cultured cell from a group ofcultured cells can be useful as a first step in the process of nucleartransfer, as described hereafter. When a cell is dissociated from anembryo, the dissociation manipulation can be useful for such processesas re-cloning, a process described herein, as well as a step formultiplying the number of embryos.

In another aspect, the invention features a totipotent mammalian cell,where the cell is immortalized, prepared by a process comprising thesteps of: (a) isolating at least one precursor cell; and (b) introducinga stimulus to the precursor cell that converts the precursor cell intothe totipotent mammalian cell.

The term “converts” as used herein refers to the phenomenon in whichprecursor cells become immortalized and/or totipotent. The term“convert” is synonymous with the term “reprogram” as used herein whenthe precursor cell is non-immortalized and/or non-totipotent. Precursorcells can be converted into totipotent, immortalized cells in varyingproportions. For example, it is possible that only a small portion ofprecursor cells are converted into totipotent, immortalized cells. Inthe art, some researchers have discussed techniques for convertingprecursor cells into pluripotent cells. Matsui et al., 1992, Cell 70:841-847.

The term “stimulus” as used herein refers to materials and/or methodsuseful for converting precursor cells into immortalized and/ortotipotent cells. The stimulus can be electrical, mechanical,temperature-related, and/or chemical, for example. The stimulus may be acombination of one or more different types of stimuli. As describedherein in exemplary embodiments, placing precursor cells in culture canbe a sufficient stimulus to convert precursor cells into immortalizedand/or totipotent cells. A stimulus can be introduced to precursor cellsfor any period of time that accomplishes the conversion of precursorcells into immortalized and/or totipotent cells.

The term “introduce” as used herein in reference to a stimulus refers toa step or steps in which precursor cells are contacted with a stimulus.If the stimulus is chemical in nature, for example, the stimulus may beintroduced to the precursor cells by mixing the stimulus with cellculture medium.

In preferred embodiments (1) the precursor cells are co-cultured withfeeder cells; (2) the precursor cells are not co-cultured with feedercells; (3) the feeder cells are established from fetal cells; (4) thefetal cells arise from a fetus where no cell types have been removedfrom the fetus; (5) the fetal cells arise from a fetus where one or morecell types have been removed from the fetus; (6) the stimulus isintroduced to precursor cells by feeder cells; (7) the feeder cells arethe only source of the stimulus; (8) the stimulus is introduced to theprecursor cells in a mechanical fashion; (9) the only stimulus isintroduced to the precursor cells in a mechanical fashion; (10) thestimulus is introduced to the precursor cells by feeder cells and in amechanical fashion; (11) the stimulus comprises the step of incubatingthe precursor cells with a receptor ligand cocktail; (12) the precursorcells are isolated from an ungulate animal and preferably a bovineanimal; (13) the precursor cells are selected from the group consistingof non-embryonic cells, primordial germ cells, genital ridge cells,amniotic cells, fetal fibroblast cells, ovarian follicular cells,cumulus cells, hepatic cells, differentiated cells, cells that originatefrom an animal, embryonic stem cells, fetal cells, and embryonic cells;(14) the receptor ligand cocktail comprises at least one componentselected from the group consisting of cytokine, growth factor, trophicfactor, and neurotrophic factor, LIF, and FGF; (15) the LIF has an aminoacid sequence substantially similar to the amino acid sequence of humanLIF; and (16) the FGF has an amino acid sequence substantially similarto the amino acid sequence of bovine bFGF.

The terms “mechanical fashion” and “mechanical stimulus” as used hereinrefers to introducing a stimulus to cells where the stimulus is notintroduced by feeder cells. For example, purified LIF and bFGF (definedhereafter) can be introduced as a stimulus to precursor cells by addingthese purified products to a cell culture medium in which the precursorcells are growing.

The term “feeder cells” as used herein refers to cells grown inco-culture with target cells. Target cells can be precursor cells andtotipotent cells, for example. Feeder cells can provide, for example,peptides, polypeptides, electrical signals, organic molecules (e.g.,steroids), nucleic acid molecules, growth factors (e.g., bFGF), otherfactors (e.g., cytokines such as LIF and steel factor), and metabolicnutrients to target cells. Certain cells, such as immortalized,totipotent cells may not require feeder cells for healthy growth. Feedercells preferably grow in a mono-layer.

Feeder cells can be established from multiple cell types. Examples ofthese cell types are fetal cells, mouse cells, Buffalo rat liver cells,and oviductal cells. These examples are not meant to be limiting. Tissuesamples can be broken down to establish a feeder cell line by methodswell known in the art (e.g., by using a blender). Feeder cells mayoriginate from the same or different animal species as the precursorcells. In an example of feeder cells established from fetal cells,ungulate fetuses and preferably bovine fetuses may be utilized toestablish a feeder cell line where one or more cell types have beenremoved from the fetus (e.g., primordial germs cells, cells in the headregion, and cells in the body cavity region). When an entire fetus isutilized to establish a fetal feeder cell line, feeder cells (e.g.,fibroblast cells) and precursor cells (e.g., primordial germ cells) canarise from the same source (e.g., one fetus).

The term “receptor ligand cocktail” as used herein refers to a mixtureof one or more receptor ligands. A receptor ligand refers to anymolecule that binds to a receptor protein located on the outside or theinside of a cell. Receptor ligands can be selected from molecules of thecytokine family of ligands, neurotrophin family of ligands, growthfactor family of ligands, and mitogen family of ligands, all of whichare well known to a person of ordinary skill in the art. Examples ofreceptor/ligand pairs are: epidermal growth factor receptor/epidermalgrowth factor, insulin/insulin receptor, cAMP-dependent proteinkinase/cAMP, growth hormone receptor/growth hormone, and steroidreceptor/steroid. It has been shown that certain receptors exhibitcross-reactivity. For example, heterologous receptors, such asinsulin-like growth factor receptor 1 (IGFR1) and insulin-like growthfactor receptor 2 (IGFR2) can both bind IGF1. When a receptor ligandcocktail comprises the stimulus, the receptor ligand cocktail can beintroduced to the precursor cell in a variety of manners known to aperson of ordinary skill in the art.

The term “cytokine” as used herein refers to a large family of receptorligands well-known to a person of ordinary skill in the art. Thecytokine family of receptor ligands includes such members as leukemiainhibitor factor (LIF), cardiotrophin 1 (CT-1), ciliary neurotrophicfactor (CNTF), stem cell factor (SCF), oncostatin M (OSM), and anymember of the interleukin (IL) family, including IL-6, IL-11, and IL-12.The teachings of the invention do not require the mechanical addition ofsteel factor (also known as stem cell factor in the art) for theconversion of precursor cells into totipotent cells.

The term “growth factor” as used herein refers to any receptor ligandthat causes a cell growth and/or cell proliferation effect. Examples ofgrowth factors are well known in the art. Fibroblast growth factor (FGF)is one example of a growth factor. The term “bFGF” can refer to basicFGF.

The term “substantially similar” as used herein in reference to aminoacid sequences refers to two amino acid sequences having preferably 50%or more amino acid identity, more preferably 70% or more amino acididentity or most preferably 90% or more amino acid identity. Amino acididentity is a property of amino acid sequence that measures theirsimilarity or relationship. Identity is measured by dividing the numberof identical residues in the two sequences by the total number ofresidues and multiplying the product by 100. Thus, two copies of exactlythe same sequence have 100% identity, while sequences that are lesshighly conserved and have deletions, additions, or replacements have alower degree of identity. Those of ordinary skill in the art willrecognize that several computer programs are available for performingsequence comparisons and determining sequence identity.

In another aspect, the invention features a method for preparing atotipotent mammalian cell, where the cell is immortalized, comprisingthe following steps: (a) isolating one or more precursor cells; and (b)introducing the precursor cell to a stimulus that converts the precursorcell into the totipotent cell. Any of the embodiments defined previouslyherein in reference to totipotent mammalian cells relate to the methodfor preparing a totipotent mammalian cell.

Cloned Totipotent Embryos of the Invention

The invention relates in part to cloned totipotent embryos. Hence,aspects of the invention feature cloned mammalian embryos where (1) theembryo is totipotent; (2) the embryo arises from an immortalized and/ortotipotent cell; and (3) the embryo arises from a non-embryonic cell;and (4) any combination of the foregoing.

The term “totipotent” as used herein in reference to embryos refers toembryos that can develop into a live born animal. The term “live born”is defined previously.

The term “cloned” as used herein refers to a cell, embryonic cell, fetalcell, and/or animal cell having a nuclear DNA sequence that issubstantially similar or identical to the nuclear DNA sequence ofanother cell, embryonic cell, fetal cell, and/or animal cell. The terms“substantially similar” and “identical” are described herein. The clonedembryo can arise from one nuclear transfer, or alternatively, the clonedembryo can arise from a cloning process that includes at least onere-cloning step. If the cloned embryo arises from a cloning procedurethat includes at least one re-cloning step, then the cloned embryo canindirectly arise from an immortalized, totipotent cell since there-cloning step can utilize embryonic cells isolated from an embryo thatarose from an immortalized, totipotent cell.

In preferred embodiments, (1) the cloned mammalian embryo is preferablyan ungulate embryo and more preferably a bovine embryo; (2) the clonedbovine embryo can be one member of a plurality of embryos, where theplurality of embryos share a substantially similar nuclear DNA sequence;(3) the cloned mammalian embryo can be one member of a plurality ofembryos, and the plurality of embryos can have an identical nuclear DNAsequence; (4) the cloned mammalian embryo has a nuclear DNA sequencethat is substantially similar to a nuclear DNA sequence of a live bornmammalian animal; (5) one or more cells of the cloned mammalian embryohave modified nuclear DNA; (6) the cloned mammalian embryo is subject tomanipulation; (7) the manipulation comprises the step of culturing theembryo in a suitable medium; (8) the suitable medium for culturing theembryo is CR-2 medium; (9) the medium can comprise feeder cells; (10)the manipulation of an embryo comprises the step of implanting theembryo into the uterus of a female; (11) the female animal is preferablyan ungulate animal and more preferably a bovine animal; (12) the estruscycle of the female is synchronized with the development cycle of theembryo; and (13) the manipulation comprises the step of incubating theembryo in an artificial environment.

All preferred embodiments related to modified nuclear DNA for totipotentcells of the invention extend to cloned embryos of the invention. Inaddition, any of the manipulations described in conjunction withtotipotent cells of the invention apply to cloned embryos of theinvention.

The term “substantially similar” as used herein in reference to nuclearDNA sequences refers to two nuclear DNA sequences that are nearlyidentical. The two sequences may differ by copy error differences thatnormally occur during the replication of a nuclear DNA. Substantiallysimilar DNA sequences are preferably greater than 97% identical,more-preferably greater than 98% identical, and most preferably greaterthan 99% identical. The term “identity” as used herein in reference tonuclear DNA sequences can refer to the same usage of the term inreference to amino acid sequences, which is described previously herein.

The term “plurality” as used herein in reference to embryos refers to aset comprising at least two embryos having a substantially similarnuclear DNA sequence. In preferred embodiments, the plurality consistsof five or more embryos, ten or more embryos, one-hundred or moreembryos, or one-thousand or more embryos. Because the occurrence of morethan three embryos progressing to term only occurs with a probability ofapproximately 1/100,000, a plurality of at least five embryos or animalsrelates to cloned embryos or cloned animals rather than naturallyoccurring embryos or animals.

The term “culturing” as used herein with respect to embryos refers tolaboratory procedures that involve placing an embryo in a culturemedium. The embryo can be placed in the culture medium for anappropriate amount of time to allow the embryo to remain static butfunctional in the medium, or to allow the embryo to grow in the medium.Culture media suitable for culturing embryos are well-known to thoseskilled in the art. See, e.g., U.S. Pat. No. 5,213,979, entitled “Invitro Culture of Bovine Embryos,” First et al., issued May 25, 1993, andU.S. Pat. No. 5,096,822, entitled “Bovine Embryo Medium,” Rosenkrans,Jr. et al., issued Mar. 17, 1992, incorporated herein by reference intheir entireties including all figures, tables, and drawings.

The term “suitable medium” as used herein refers to any medium thatallows cell proliferation. The suitable medium need not promote maximumproliferation, only measurable cell proliferation. A suitable medium forembryo development is discussed previously.

The term “CR-2 medium” as used herein refers to a medium suitable forculturing embryos. CR-2 medium can comprise one or more of the followingcomponents: sodium chloride; potassium chloride; sodium bicarbonate;hemicalcium L-lactate; and fatty-acid free BSA. These components mayexist in the medium in concentrations of about 115 mM for sodiumchloride; about 3 mM for potassium chloride; about 25 mM for sodiumbicarbonate; about 5 mM for hemicalcium L-lactate; and about 3 mg/mL forfatty-acid free BSA. Alternatively, the concentrations of thesecomponents may exist in the medium in concentrations of 0-1 M sodiumchloride; 0-100 mM potassium chloride; 0-500 mM sodium bicarbonate;0-100 mM hemicalcium L-lactate; and 0-100 mg/mL fatty-acid free BSA.

The term “feeder cells” is defined previously herein. Embryos of theinvention can be cultured in media with or without feeder cells. Inother preferred embodiments, the feeder cells can be cumulus cells.

The term “implanting” as used herein in reference to embryos refers toimpregnating a female animal with an embryo described herein. Thistechnique is well known to a person of ordinary skill in the art. See,e.g., Seidel and Elsden, 1997, Embryo Transfer in Dairy Cattle, W. D.Hoard & Sons, Co., Hoards Dairyman. The embryo may be allowed to developin utero, or alternatively, the fetus may be removed from the uterineenvironment before parturition.

The term “synchronized” as used herein in reference to estrus cycle,refers to assisted reproductive techniques well known to a person ofordinary skill in the art. These techniques are fully described in thereference cited in the previous paragraph. Typically, estrogen andprogesterone hormones are utilized to synchronize the estrus cycle ofthe female animal with the developmental cycle of the embryo. The term“developmental cycle” as used herein refers to embryos of the inventionand the time period that exists between each cell division within theembryo. This time period is predictable for embryos from ungulates, andcan be synchronized with the estrus cycle of a recipient animal.

The term “artificial environment” refers to one that promotes thedevelopment of an embryo or other developing cell mass. An artificialenvironment can be a uterine environment or an oviductal environment ofa species different from that of the developing cell mass. For example,a developing bovine embryo can be placed into the uterus or oviduct ofan ovine animal. Stice & Keefer, 1993, “Multiple generational bovineembryo cloning,” Biology of Reproduction 48: 715-719. Alternatively, anartificial development environment can be assembled in vitro. This typeof artificial uterine environment can be synthesized using biologicaland chemical components known in the art.

In another aspect the invention features a cloned mammalian embryo,where the embryo is totipotent, prepared by a process comprising thestep of nuclear transfer. Preferably, nuclear transfer occurs between(a) a totipotent mammalian cell, where the cell is immortalized, and (b)an oocyte, where the oocyte is at a stage allowing formation of theembryo.

In preferred embodiments, (1) the oocyte is an enucleated oocyte; (2)the totipotent mammalian cell and the oocyte preferably originate froman ungulate animal and more preferably originate from a bovine animal;(3) the totipotent mammalian cell can originate from one specie ofungulate and the oocyte can originate from another specie of ungulate;(4) the oocyte is a young oocyte; (5) the totipotent mammalian cell isplaced in the perivitelline space of the oocyte; (6) the totipotent cellutilized for nuclear transfer can arise from any of the cells describedpreviously (e.g., a non-embryonic cell, a primordial germ cell, agenital ridge cell, a differentiated cell, a fetal cell, a non-fetalcell, a non-primordial germ cell, an amniotic cell, a fetal fibroblastcell, an ovarian follicular cell, a cumulus cell, an hepatic cell, acell isolated from an asynchronous population of cells, a cell isolatedfrom a synchronous population of cells, a cell isolated from an existinganimal, and an embryonic stem cell); (7) the nuclear transfer comprisesthe step of translocation of the totipotent mammalian-cell into therecipient oocyte; (8) the translocation can comprise the step ofinjection of the totipotent mammalian cell nuclear donor into therecipient oocyte; (9) the translocation can comprise the step of fusionof the totipotent mammalian cell and the oocyte; (10) the fusion cancomprise the step of delivering one or more electrical pulses to thetotipotent mammalian cell and the oocyte; (11) the fusion can comprisethe step of delivering a suitable concentration of at least one fusionagent to the totipotent mammalian cell and the oocyte; (12) the nucleartransfer may comprise the step of activation of the totipotent mammaliancell and the oocyte; and (13) the activation is accomplished byintroducing DMAP and/or ionomycin to an oocyte and/or a cybrid.

The term “enucleated oocyte” as used herein refers to an oocyte whichhas had part of its contents removed. Typically a needle can be placedinto an oocyte and the nucleus can be aspirated into the inner space ofthe needle. The needle can be removed from the oocyte without rupturingthe plasma membrane. This enucleation technique is well known to aperson of ordinary skill in the art. See, U.S. Pat. Nos. 4,994,384;5,057,420; and Willadsen, 1986, Nature 320:63-65. An enucleated oocytecan be prepared from a young or an aged oocyte. Definitions of “youngoocyte” and “aged oocyte” are provided herein. Nuclear transfer may beaccomplished by combining one nuclear donor and more than one enucleatedoocyte. In addition, nuclear transfer may be accomplished by combiningone nuclear donor, one or more enucleated oocytes, and the cytoplasm ofone or more enucleated oocytes.

The term “cybrid” as used herein refers to a construction where anentire nuclear donor is translocated into the cytoplasm of a recipientoocyte. See, e.g., In Vitro Cell. Dev. Biol. 26: 97-101 (1990).

The invention specifically relates to cloned mammalian embryos createdby nuclear transfer, where the nucleus of the oocyte is not physicallyextracted from the nucleus. It is possible to create a cloned embryowhere the nuclear DNA from the donor cell is the material replicatedduring cellular divisions. See, e.g. Wagoner et al., 1996, “Functionalenucleation of bovine oocytes: effects of centrifugation and ultravioletlight,” Theriogenology 46: 279-284.

The term “another ungulate” as used herein refers to a situation wherethe nuclear donor originates from an ungulate of a different species,genera or family than the ungulate from which the recipient oocyteoriginates. For example, the totipotent mammalian cell used as a nucleardonor can arise from a water buffalo, while the oocyte recipient canarise from a domestic cow. This example is not meant to be limiting andany ungulate species/family combination of nuclear donors and recipientoocytes are foreseen by the invention.

The term “young oocyte” as used herein refers to an oocyte that has beenmatured in vitro and/or ovulated in vivo for less than 28 hours sincethe onset of maturation. Oocytes can be isolated from live animals usingmethods well known to a person of ordinary skill in the art. See, e.g.,Pieterse et al., 1988, “Aspiration of bovine oocytes during transvaginalultrasound scanning of the ovaries,” Theriogenology 30: 751-762. Oocytescan be isolated from ovaries or oviducts or deceased or live bornanimals. Suitable media for in vitro culture of oocytes are well knownto a person of ordinary skill in the art. See, e.g., U.S. Pat. No.5,057,420, which is incorporated by reference herein.

The term “maturation” as used herein refers to process in which anoocyte is incubated in a medium in vitro. Oocytes can be incubated withmultiple media well known to a person of ordinary skill in the art. See,e.g., Saito et al., 1992, Roux's Arch. Dev. Biol. 201: 134-141 forbovine organisms and Wells et al., 1997, Biol. Repr. 57: 385-393 forovine organisms, both of which are incorporated herein by reference intheir entireties including all figures, tables, and drawings. Maturationmedia can comprise multiple types of components, including microtubuleinhibitors (e.g., cytochalasin B). Other examples of components that canbe incorporated into maturation media are discussed in WO 97/07668,entitled “Unactivated Oocytes as Cytoplast Recipients for NuclearTransfer,” Campbell & Wilmut, published on Mar. 6, 1997, herebyincorporated herein by reference in its entirety, including all figures,tables, and drawings. The time of maturation can be determined from thetime that an oocyte is placed in a maturation medium and the time thatthe oocyte is then utilized in a nuclear transfer procedure.

Young oocytes can be identified by the appearance of their ooplasm.Because certain cellular material (e.g., lipids) have not yet dispersedwithin the ooplasm. Young oocytes can have a pycnotic appearance. Apycnotic appearance can be characterized as clumping of cytoplasmicmaterial. A “pycnotic” appearance is to be contrasted with theappearance of oocytes that are older than 28 hours, which have a morehomogenous appearing ooplasm.

The term “translocation” as used herein in reference to nuclear transferrefers to the combining of a totipotent mammalian cell nuclear donor anda recipient oocyte. The translocation may be performed by suchtechniques as fusion and/or direct injection, for example.

The term “injection” as used herein in reference to embryos, refers tothe perforation of the oocyte with a needle, and insertion of thenuclear donor in the needle into the oocyte. In preferred embodiments,the nuclear donor may be. injected into the cytoplasm of the oocyte orin the perivitelline space of the oocyte. This direct injection approachis well known to a person of ordinary skill in the art, as indicated bythe publications already incorporated herein in reference to nucleartransfer. For the direct injection approach to nuclear transfer, thewhole totipotent mammalian cell may be injected into the oocyte, oralternatively, a nucleus isolated from the totipotent mammalian cell maybe injected into the oocyte. Such an isolated nucleus may be surroundedby nuclear membrane only, or the isolated nucleus may be surrounded bynuclear membrane and plasma membrane in any proportion. The oocyte maybe pre-treated to enhance the strength of its plasma membrane, such asby incubating the oocyte in sucrose prior to injection of the nucleardonor.

Techniques for placing a nuclear donor (e.g., an immortalized andtotipotent cell of the invention) into the perivitelline space of anenucleated oocyte are well known to a person of ordinary skill in theart, and are fully described in the patents and references citedpreviously herein in reference to nuclear transfer.

The term “fusion” as used herein refers to the combination of portionsof lipid membranes corresponding to the totipotent mammalian cellnuclear donor and the recipient oocyte. Lipid membranes can correspondto the plasma membranes of cells or nuclear membranes, for example. Thefusion can occur between the nuclear donor and recipient oocyte whenthey are placed adjacent to one another, or when the nuclear donor isplaced in the perivitelline space of the recipient oocyte, for example.Specific examples for translocation of the totipotent mammalian cellinto the oocyte are described hereafter in other preferred embodiments.These techniques for translocation are fully described in the referencescited previously herein in reference to nuclear transfer.

The term “electrical pulses” as used herein refers to subjecting thenuclear donor and recipient oocyte to electric current. For nucleartransfer, the nuclear. donor and recipient oocyte can be aligned betweenelectrodes and subjected to electrical current. The electrical currentcan be alternating current or direct current. The electrical current canbe delivered to cells for a variety of different times as one pulse oras multiple pulses. The cells are typically cultured in a suitablemedium for the delivery of electrical pulses. Examples of electricalpulse conditions utilized for nuclear transfer are described in thereferences and patents previously cited herein in reference to nucleartransfer.

The term “fusion agent” as used herein refers to any compound orbiological organism that can increase the probability that portions ofplasma membranes from different cells will fuse when a totipotentmammalian cell nuclear donor is placed adjacent to the recipient oocyte.In preferred embodiments fusion agents are selected from the groupconsisting of polyethylene glycol (PEG), trypsin, dimethylsulfoxide(DMSO), lectins, agglutinin, viruses, and Sendai virus. These examplesare not meant to be limiting and other fusion agents known in the artare applicable and included herein.

The term “suitable concentration” as used herein in reference to fusionagents, refers to any concentration of a fusion agent that affords ameasurable amount of fusion. Fusion can be measured by multipletechniques well known to a person of ordinary skill in the art, such asby utilizing a light microscope, dyes, and fluorescent lipids, forexample.

The term “activation” refers to any materials and methods useful forstimulating a cell to divide before, during, and after a nucleartransfer step. Cybrids may require stimulation in order to divide aftera nuclear transfer has occurred. The invention pertains to anyactivation materials and methods known to a person of ordinary skill inthe art. Although electrical pulses are sometimes sufficient forstimulating activation of cybrids, other means are sometimes useful ornecessary for proper activation of the cybrid. Chemical materials andmethods useful for activating embryos are described below in otherpreferred embodiments of the invention.

Examples of non-electrical means for activation include agents such asethanol; inositol trisphosphate (IP₃); Ca⁺⁺ ionophores (e.g., ionomycin)and protein kinase inhibitors (e.g., 6-dimethylaminopurine (DMAP));temperature change; protein synthesis inhibitors (e.g., cyclohexamide);phorbol esters such as phorbol 12-myristate 13-acetate (PMA); mechanicaltechniques; and thapsigargin. The invention includes any activationtechniques known in the art. See, e.g., U.S. Pat. No. 5,496,720,entitled “Parthenogenic Oocyte Activation,” issued on Mar. 5, 1996,Susko-Parrish et al., incorporated by reference herein in its entirety,including all figures, tables, and drawings.

In other preferred embodiments, (1) one or more cells of the clonedembryo comprise modified nuclear DNA; (2) the cloned embryo is subjectto manipulation; (3) the manipulation comprises the step ofdisaggregating at least one individual cell from a cloned embryo; (4)the manipulation comprises the step of utilizing the individual cell asa nuclear donor in a nuclear transfer procedure; (5) the individual cellis disaggregated from the inner cell mass of a blastocyst stage embryo;(6) the individual cell is disaggregated from a pre-blastocyst stageembryo; (7) the manipulation comprises the process of re-cloning; (8)the re-cloning process comprises the steps of: (a) separating the embryointo one or more individual cells, and (b) performing at least onesubsequent nuclear transfer between (i) an individual cell of (a), and(ii) an oocyte; (9) the oocyte utilized for the subsequent nucleartransfer is an aged oocyte; (10) the individual cell is placed in theperivitelline space of the enucleated oocyte for the subsequent nucleartransfer; (11) the subsequent nuclear transfer comprises at least one ofthe steps of translocation, injection, fusion, and activation of theindividual cell and/or the enucleated oocyte; (12) one or more cells ofthe cloned mammalian embryo arising from the subsequent nuclear transfercomprises modified nuclear DNA; and (13) the cloned mammalian embryoarising from the subsequent nuclear transfer may be'subject to asubsequent manipulation, where the subsequent manipulation is any of themanipulation steps defined previously herein in relation to immortalizedcells and/or cloned embryos.

The term “individual cells” as used herein refers to cells that havebeen isolated from a cloned mammalian embryo of the invention. Anindividual single cell can be isolated from the rest of the embryonicmass by techniques well known to those skilled in the art. See, U.S.Pat. Nos. 4,994,384 and 5,957,420, previously incorporated herein byreference in their entireties.

The term “subsequent nuclear transfer” as described herein is alsoreferred to as a “re-cloning” step. Preferably, a re-cloning step can beutilized to enhance nuclear reprogramming during nuclear transfer, suchthat the product of nuclear transfer is a live born animal. There-cloning step is distinct, since previous efforts towards re-cloninghave been directed to multiplying embryo number and not for enhancementof nuclear reprogramming. The number of subsequent nuclear transfersteps is discussed in greater detail hereafter.

Any of the preferred embodiments related to the translocation,injection, fusion, and activation steps described previously hereinrelate to the subsequent nuclear transfer step.

The term “inner cell mass” as used herein refers to the cells that givesrise to the embryo proper. The cells that line the outside of ablastocyst are referred to as the trophoblast of the embryo. The methodsfor isolating inner cell mass cells from an embryo are well known to aperson of ordinary skill in the art. See, Sims and First, 1993,Theriogenology 39:313; and Keefer et al., 1994, Mol. Reprod. Dev.38:264-268, hereby incorporated by reference herein in their entireties,including all figures, tables, and drawings. The term “pre-blastocyst”is well known in the art and is referred to previously.

The term “aged oocyte” as used herein refers to an oocyte that has beenmatured in vitro or ovulated in vivo for more than 28 hours since theonset of maturation or ovulation. An aged oocyte can be identified byits characteristically homogenous ooplasm. This appearance is to becontrasted with the pycnotic appearance of young oocytes as describedpreviously herein. The age of the oocyte can be defined by the time thathas elapsed between the time that the oocyte is placed in a suitablematuration medium and the time that the oocyte is activated. The age ofthe oocyte can dramatically enhance the efficiency of nuclear transfer.

The term “ovulated in vivo” as used herein refers to an oocyte that isisolated from an animal a certain number of hours after the animalexhibits characteristics that it is in estrus. The characteristics of ananimal in estrus are well known to a person of ordinary skill in theart, as described in references disclosed herein.

In another aspect the invention relates to a method for preparing acloned mammalian embryo. The method comprises the step of a nucleartransfer between: (a) a totipotent mammalian cell, where the cell isimmortalized; and (b) an oocyte, where the oocyte is at a stage allowingformation of the embryo. In preferred embodiments, any of theembodiments of the invention concerning cloned mammalian embryos applyto methods for preparing cloned mammalian embryos.

Cloned Fetuses of the Invention

In another aspect, the invention features cloned mammalian fetusesarising from totipotent embryos of the invention. Preferably, themammalian fetuses are ungulate fetuses, and more preferably, theungulate fetuses are bovine fetuses. A fetus may be isolated from theuterus of a pregnant female animal.

In preferred embodiments, (1) one or more cells of the fetuses harbormodified nuclear DNA (defined previously herein); and (2) the fetusesmay be subject to any of the manipulations defined herein. For example,one manipulation may comprise the steps of isolating a fetus from theuterus of a pregnant female animal, isolating a cell from the fetus(e.g., a primordial germ cell), and utilizing the isolated cell as anuclear donor for nuclear transfer.

Other aspects of the invention feature (1) a cloned mammalian fetusprepared by a process comprising the steps of (a) preparation of acloned mammalian embryo defined previously, and (b) manipulation of thecloned mammalian embryo such that it develops into a fetus; (2) a methodfor preparing a cloned mammalian fetus comprising the steps of (a)preparation of a cloned mammalian embryo defined previously, and (b)manipulation of the cloned mammalian embryo such that it develops into afetus; (3) a method of using a cloned fetus of the invention comprisingthe step of isolating at least one cell type from a fetus (e.g., forcreating a feeder cell layer); and (4) a method of using a cloned fetusof the invention comprising the step of separating at least one part ofa fetus into individual cells (e.g., for establishing a feeder celllayer).

Cloned Animals of the Invention

In another aspect the invention features a cloned mammalian animalarising from a cloned embryo of the invention. The embryo is totipotentand can arise from any of the processes or methods described previouslyherein.

In preferred embodiments, the cloned mammalian animal (1) is preferablya cloned ungulate animal and more preferably a cloned bovine animal; and(2) is equal in age or older than an animal selected from the groupconsisting of pre- and post-pubertal animals.

In yet another aspect the invention relates to a cloned mammaliananimal, where the animal is one member of a plurality of animals, andwhere the plurality of animals have a substantially similar nuclear DNAsequence. The term “substantially similar” in relation to nuclear DNAsequences is defined previously herein.

In preferred embodiments, (1) the plurality consists of five or moreanimals, ten or more animals, one-hundred or more animals, andone-thousand or more animals; and (2) the plurality of animals can havean identical nuclear DNA sequence. The term “identical” in reference tonuclear DNA sequences is described previously herein.

In another aspect, the invention relates to a cloned mammalian animalhaving one or more cells that comprise modified nuclear DNA. All of thepreferred embodiments relating to modified nuclear DNA describedpreviously apply to cloned bovine animals of the invention.

In yet another aspect, the invention features a method of using a clonedmammalian animal, comprising the step of isolating at least onecomponent from the mammalian animal.

The term “component” as used herein refers to any portion of an animal.A component can be selected from the group consisting of fluid,biological fluid, cell, tissue, organ, gamete, embryo, and fetus.Precursor cells may arise from fluids, biological fluids, cells,tissues, organs, gametes, embryos, and fetuses isolated from clonedorganisms of the invention.

The term “gamete” as used herein refers to any cell participating,directly or indirectly, to the reproductive system of an animal.Examples of gametes are spermatocytes, spermatogoria, oocytes, andoogonia. Gametes can be present in fluids, tissues, and organs collectedfrom animals (e.g., sperm is present in semen). For example, methods ofcollecting semen for the purposes of artificial insemination are wellknown to a person of ordinary skill in the art. See, e.g., Physiology ofReproduction and Artificial Insemination of Cattle (2nd edition),Salisbury et al., copyright 1961, 1978, W H Freeman & Co., SanFrancisco. However, the invention relates to the collection of any typeof gamete from an animal.

The term “tissue” is defined previously. The term “organ” refers to anyorgan isolated from an animal or any portion of an organ. Examples oforgans and tissues are neuronal tissue, brain tissue, spleen, heart,lung, gallbladder, pancreas, testis, ovary and kidney. These examplesare not limiting and the invention relates to any organ and any tissueisolated from a cloned animal of the invention.

In a preferred embodiments, the invention relates to (1) fluids,biological fluids, cells, tissues, organs, gametes, embryos, and fetusescan be subject to manipulation; (2) the manipulation can comprise thestep of cryopreserving the gametes, embryos, and/or fetal tissues; (3)the manipulation can comprise the step of thawing the cryopreserveditems; (4) the manipulation can comprise the step of separating thesemen into X-chromosome bearing semen and Y-chromosome bearing semen;(5) the manipulation comprises methods of preparing the semen forartificial insemination; (6) the manipulation comprises the step ofpurification of a desired polypeptide(s) from the biological fluid ortissue; (7) the manipulation comprises concentration of the biologicalfluids or tissues; and (8) the manipulation can comprise the step oftransferring one or more cloned cells, cloned tissues, cloned organs,and/or portions of cloned organs to a recipient organism (e.g., therecipient organism may be of a different specie than the donor source).

The term “separating” as used herein in reference to separating semenrefers to methods well known to a person skilled in the art forfractionating a semen sample into sex-specific fractions. This type ofseparation can be accomplished by using flow cytometers that arecommercially available. Methods of utilizing flow cytometers fromseparating sperm by genetic content are well known in the art. Inaddition, semen can be separated by its sex-associated characteristicsby other methods well known to a person of ordinary skill in the art.See, U.S. Pat. Nos. 5,439,362, 5,346,990, and 5,021,244, entitled“Sex-Associated Membrane Proteins and Methods for Increasing theProbability that Offspring Will Be of a Desired Sex,” Spaulding, issuedon Aug. 8, 1995, Sep. 13, 1994, and Jun. 4, 1991 respectively, all ofwhich are incorporated herein by reference in their entireties includingall figures, tables, and drawings.

Semen preparation methods are well known to someone of ordinary skill inthe art. Examples of these preparative steps are described in Physiologyof Reproduction and Artificial Insemination of Cattle (2nd. edition),Salisbury et al., copyright 1961, 1978, W.H. Freeman & Co., SanFrancisco.

The term “purification” as used herein refers to increasing the specificactivity of a particular polypeptide or polypeptides in a sample. Inpreferred embodiments, specific activity is expressed as the ratiobetween the activity of the target polypeptide and the concentration oftotal polypeptide in the sample. Activity can be catalytic activityand/or binding activity, for example. In other preferred embodiments,specific activity is expressed as the ratio between the concentration ofthe target polypeptide and the concentration of total polypeptide.Purification methods include dialysis, centrifugation, and columnchromatography techniques, which are well-known procedures to a personof ordinary skill in the art. See, e.g., Young et al., 1997, “Productionof biopharmaceutical proteins in the milk of transgenic dairy animals,”BioPharm 10(6): 34-38.

The term “transferring” as used herein refers to shifting a group ofcells, tissues, organs, and/or portions of organs to an animal. Thecells, tissues, organs, and/or portions of organs can be, for example,(a) developed in vitro and then transferred to an animal, (b) removedfrom an animal and transferred to another animal of a different specie,(c) removed from an animal and transferred to another animal of the samespecie, (d) removed from one portion of an animal (e.g., the leg of ananimal) and then transferred to another portion of the same animal (e.g.the brain of the animal), and/or (e) any combination of the foregoing.The term “transferring” refers to adding cells, tissues, and/or organsto an animal and can also relate to removing cells, tissues, and/ororgans from an animal and replacing them with cells, tissues, and/ororgans from another source.

The term “transferring” as used herein also refers to implanting one ormore cells, tissues, organs, and/or portions of organs from the clonedmammalian animal into another organism. For example, neuronal tissuefrom a cloned mammalian organism can be grafted into an appropriate areain the human nervous system to treat neurological diseases such asAlzheimer's disease. Alternatively, cloned cells, tissues, and/or organsoriginating from a porcine organism may be transferred to a humanrecipient. Surgical methods for accomplishing this preferred aspect ofthe invention are well known to a person of ordinary skill in the art.Transferring procedures may include the step of removing cells, tissues,or organs from a recipient organism before a transfer step.

In other aspects the invention features (1) a cloned mammalian animalprepared by a process comprising the steps of: (a) preparation of acloned mammalian embryo by any one of the methods described herein forproducing such a cloned mammalian embryo, and (b) manipulation of thecloned mammalian embryo such that it develops into a live born animal;(2) a process comprising the steps of: (a) preparation of a clonedmammalian embryo by any one of the methods described herein forpreparing such a cloned mammalian embryo, and (b) manipulation of thecloned mammalian embryo such that it develops into a live born animal;and (3) a cloned mammalian animal, comprising the steps of: (a)preparation of a cloned mammalian embryo by any one of the methods forproducing such an embryo described herein, and (b) manipulation of thecloned mammalian embryo such that it develops into a live born animal.

In preferred embodiments, (1) the live born animal is preferably anungulate animal and more preferably a bovine animal; (2) themanipulation can comprise the step of implanting the embryo into auterus of an animal; (3) the estrus cycle of the animal can besynchronized to the developmental stage of the embryo; and (4) themanipulation can comprise the step of implanting the embryo into anartificial environment.

The summary of the invention described above is not limiting and otherfeatures and advantages of the invention will be apparent from thefollowing detailed description of the preferred embodiments, as well asfrom the claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates multiple embodiments of the invention relating to thegeneration of immortalized, totipotent cells from precursor cells. Thefigure indicates that immortalized, totipotent cells can arise fromembryonic stem cells, primordial germ cells, and cells isolated from ananimal. The precursor cell sources illustrated by FIG. 1 are notlimiting and other precursor cell sources are described herein.

FIGS. 2(a-e) illustrates an embodiment of the invention related tocloning. The figure illustrates a cloning procedure in which (a) aprecursor cell is reprogrammed into an immortalized, totipotent cell;(b) the immortalized, totipotent cell is utilized as a donor for a firstnuclear transfer, which utilizes a young oocyte; (c) the embryo arisingfrom the first nuclear transfer is cultured; (d) a cell isolated fromthe embryo arising from the first nuclear transfer is utilized as anuclear donor for a second nuclear transfer, which utilizes an agedoocyte; and (e) the embryo resulting from the second nuclear transfermay be cultured and then allowed to develop into a live born animal. Theembryos resulting from the nuclear transfers may be cultured and/orcryopreserved and thawed.

FIG. 3 illustrates multiple embodiments of the invention related topathways for establishing totipotent cell lines and cloned animals.Fibroblast cells can be isolated from any source described herein. Thisfigure is described in further detail in the Examples section.

FIG. 4 illustrates multiple embodiments of the invention for creatingcloned transgenic cell lines and cloned transgenic animals. This figureis described in further detail in the Examples section.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention relates to cloning technologies. The presentinvention provides multiple advantages over the tools and methodscurrently utilized in the field of cloning technology. For example, theinvention relates in part to immortalized, totipotent cells useful forcloning animals. These immortalized, totipotent cells can give rise tomethods of producing cloned animals by utilizing virtually any type ofcell. For example, cells isolated from a live born animal can bereprogrammed into immortalized, totipotent cells. This feature of theinvention provides the ability to assess the phenotype of an existinganimal and then readily establish a permanent cell line for cloning thatanimal. As described previously herein, no methods in the art haveallowed for such advantages.

In addition, the immortalized, totipotent cells of the invention allowfor creating permanent cell lines from virtually any type of cell. Thisreprogramming method is described previously herein. These permanentcell lines offer a nearly unlimited source of donor cells for nucleartransfer cloning techniques. Moreover, this feature provides theadvantage of enhancing cloning efficiency due to the lowerdifferentiation rates of these cell lines than existing cell lines usedfor cloning. For example, embryonic stem cell lines can harbor multiplecolonies of cells that are not totipotent. The totipotent, immortalizedcells of the invention harbor a higher percentage of totipotent cellsthan cell lines previously reported.

Moreover, the methods and processes for creating the immortalized,totipotent cells, totipotent cloned embryos, and cloned animals of theinvention demonstrate the enhanced cloning efficiency over cloning toolsand techniques previously reported. In particular, the totipotent,immortalized cell lines and the refined nuclear transfer techniques ofthe invention provide for this enhanced cloning efficiency. Thisenhanced efficiency satisfies a long felt need in the art.

I. Immortalized and Totipotent Cells

A. Generation of Immortalized and Totipotent Cells

Immortalized, totipotent cells of the invention can be produced fromvirtually any type of precursor cell. Preferred embodiments of theinvention relate to the following types of precursor cells: (1) embryosarising from the union of two gametes in vitro or in vivo; (2) embryonicstem cells (ESC's) arising from embryos (e.g., pre-blastocyst cells andinner cell mass cells); (3) cultured and non-cultured cells derived fromthe inner cell mass of embryos; (4) cultured and non-cultured cellsarising from a fetus; (5) primordial germ cells arising from adeveloping cell mass (e.g., genital ridge cells); (6) immortalizedcultured cells arising from. primordial germ cells, where theimmortalized cells are referred to as embryonic germ cells (EGCs) in theart; (7) cultured and non-cultured cells obtained from amniotic fluid;(8) cultured and non-cultured cells arising from an ovarian follicle(e.g., cumulus cells); (9) cultured and non-cultured cells arising froma liver (e.g., hepatocytes); and (10) cultured and non-cultured cellsisolated from an animal.

ESCs and EGCs can be readily generated from methods known in the art.See, e.g., Stice et al., 1996, Biology of Reproduction 54: 100-110,hereby incorporated by reference herein in its entirety including allfigures, tables, and drawings. See also, Strelchenko, 1996,Theriogenology 45: 130-141. ESCs have been demonstrated to give rise tofetuses, from which, primordial germ cells and EGCs can be derived.Therefore, ESCs are a nearly unlimited source for primordial germ cellsand EGCs.

Cells derived from an animal can be isolated from nearly any type oftissue. For example, an ear-punch can be taken from an animal, the cellsfrom the sample can be separated, and the separated cells can besubsequently cultured in vitro by using cell culture techniques wellknown to a person of ordinary skill in the art. Preferably, cells of theinvention are extracted from bovine animals. Examples of materials andmethods for reprogramming primary culture cells into immortalized,totipotent cells are described in exemplary embodiments hereafter.

Although exemplary embodiments of the invention are directed to bovineanimals, materials and methods of the invention can be applied to thegeneration of immortalized, totipotent cells using precursor cellsisolated from any mammal. Preferably immortalized, totipotent cells areextracted from ungulates. Examples of preferred ungulates envisioned forthe invention are described previously.

Immortalized, totipotent cells of the invention are preferably generatedfrom the examples of cells indicated in the preceding paragraph aftertreatment with a receptor ligand cocktail. Examples of receptor ligandsare well known to a person of ordinary skill in the art. Cytokinesand/or growth factors are preferred receptor ligands of the invention.See, e.g., R&D Systems Catalog, 614 McKinley Place N.E., Minneapolis,Minn. 55413. In exemplary embodiments, varying amounts of humanrecombinant leukemia inhibitory factor (hrLIF) and basic bovine.fibroblast growth factor (bFGF) can be added to the culture medium toreprogram the precursor cells into immortalized, totipotent cells.Varying concentrations of these two cytokines can be added to theculture medium, preferably in concentrations of 1-1000 ng/mL, morepreferably in concentrations between 10-500 ng/mL, and most preferablyabout 100 ng/mL. Exogenous soluble and membrane-associated forms ofsteel factor are not required for converting precursor cells intototipotent, immortalized cells.

These examples are not meant to be limiting and any cytokine orcombination of cytokines can be added or deleted from those described inexemplary embodiments described hereafter. Preferred cytokines forgenerating immortalized, totipotent cells can be selected from the groupconsisting of fibroblast growth factor (FGF), leukemia inhibitor factor(LIF), cardiotrophin 1 (CT-1), ciliary neurotrophic factor (CNTF), stemcell factor (SCF), oncostatin M (OSM), and any member of the interleukin(IL) family, including IL-6, IL-11, and IL-12.

Other cytokines and other molecules besides cytokines can be added ordeleted from the receptor ligand cocktail described in the exemplaryembodiments described hereafter to create immortalized, totipotent cellsfrom any of the cells described in the previous paragraph. Any of theconditions for generating immortalized, totipotent cells can be modifiedfrom those described herein. The ability of these modified conditions togenerate immortalized, totipotent cells can be monitored by methodsdefined in the section “Identification of Immortalized and TotipotentCells” described hereafter.

B. Culturing Immortalized and Totipotent Cells

A variety of methods for culturing cells exist in the art. See, e.g.,Culture of animal cells: a manual of basic technique (2nd. edition),Freshney, copyright 1987, Alan R. Liss, Inc., New York. Particularly thecells that are precursor cells for immortalized, totipotent cells, aswell as the immortalized, totipotent cells themselves, can be grown onfeeder layers. Examples of feeder layers are well known to a person ofordinary skill in the art, and can arise from a number of differentcells that are cultured in vitro. See, e.g., Strelchenko, 1996,Theriogenology 45: 130-141, as well as exemplary embodiments describedhereafter. However, precursor cells for immortalized, totipotent cellsas well as the immortalized, totipotent cells themselves need not begrown on feeder layers.

C. Identification of Immortalized and Totipotent Cells

Identification of Immortalized Cells

Immortalized cells can be identified as those that are not confined tothe Hayflick limit. The Hayflick limit is defined by cells that dividefor more than 60 cell divisions. Hence, cells that have divided for morethan 60 cell divisions are immortalized cells. In addition, immortalizedcells typically can be passaged at lower cell densities thannon-immortalized cells.

The materials and methods described above (e.g., culturing the cellswith cytokines) can convert non-immortalized cells into immortalizedcells. Other methods exist in the art for generating immortalized celllines from primary cells. For example, manipulating the activity oftelomerase within the cells can immortalize cells. See, e.g., U.S. Pat.No. 5,645,986, entitled “Therapy and Diagnosis of Conditions Related toTelomere Length and/or Telomerase Activity,” West et al., issued Jul. 8,1997, and hereby incorporated by reference herein in its entiretyincluding all figures, drawings, and tables.

Moreover, cellular immortality can be determined by identifying both lowmolecular weight and macromolecular markers that are specific forimmortalized cells. The existence or lack of existence of a marker canbe a determination of cell immortalization. In addition, a phenomenonassociated with a marker can be an indication of immortality. Forexample, if the marker is an enzyme, an indication of the presence ofthe enzyme and/or a certain level of catalytic activity of that enzymemay be a suitable indication that a certain cell type is immortalized.

Low molecular weight markers include specific nucleosides, lipidassociated sialic acids, polyamines, and pseudouridine. These examplesare not limiting and the invention relates to any other low molecularweight markers known in the art.

Macromolecular markers can be separated into several classes includingnucleic acid polymers, peptides, polypeptides, proteins, enzymes, growthfactors, growth factor receptors, hormones, hormone receptors,oncogenes,oncogene products, and specific glycoproteins. Macromolecularmarkers can be selected from the group consisting of extracellularproteins, membrane associated proteins, and/or intracellular proteins,which may be membrane associated or soluble. One such marker forimmortalized cells is telomerase or its associated activity, forexample. See, U.S. Pat. No. 5,645,986, supra. Other examples of markersspecific for immortalized cells can be selected from the following list:

1) Epidermal growth factor (EGF) and its receptor (EGF-R)

2) Transforming growth factor-alpha (TGF-alpha) and its receptor

3) c-erbB2 receptor tytosine kinase (HER2 product)

4) Hyaluronan receptor (probably CD44, an integral membraneglycoprotein)

5) Carcinoembryonic antigen (CEA) family of tumor markers (for exampleT1, a glycosylated protein)

6) Telomerase, a ribonucleoprotein which maintains telomere length inimmortalized cells

7) Phosphatases: placental alkaline phosphatase (PLAP), germ cellalkaline phosphatase, prostate acid phosphatase (PAS)

8) Cathepsin D (catalyzes degradation of laminin).

9) Ornithine decarboxylase (ODC) (catalyzes the rate-limiting step inpolyamine synthesis)

10) Beta-glucuronidase

11) Alpha-6 integrin

12) Keratin K8

13) Oncogene products: ras oncogenes (k-ras, Ha-ras, p21), v-src, c-myc

14) Cyclin D1, cyclin A, and Retinoblastoma Gene Protein (Rb)

15) Changes in p53 expression or p53 mutations

16) Heterogeneous ribonucleoprotein-A2 (hnRNP-A2) overexpression

17) L-plastin

18) Ganglioside fucosyl-GM1

19) Mob-1 expression (mob-1) (homology to proinflammatory cytokines)

These examples are not limiting and the invention relates to any markersspecific for immortalized cells that are known in the art.

In addition to markers for immortalization known in the art, markers forimmortalization can be identified using methods well known in the art.For example, immortalization markers can be identified by analyzingparticular molecules (e.g., nucleic acid molecules and polypeptidemolecules) that are unique to specific cell types.

In examples pertaining to nucleic acid immortalization markers,immortalized and non-immortalized cells may be subjected to analysis fornucleic acid sequence content (e.g., hybridization techniques withnucleic acid probes). Nucleic acid samples from particular immortalizedcells and nucleic acid samples from particular non-immortalized cellscan be screened for particular nucleic acid sequences. If samples fromnon-immortalized cells lack a nucleic acid sequence present inimmortalized cells, then this nucleic acid sequence could be a markerfor distinguishing immortalized cells from non-immortalized cells.Similarly, if samples from non-immortalized cells harbor a nucleic acidsequence that immortalized cells lack, this nucleic acid sequence couldbe a marker for distinguishing immortalized cells from non-immortalizedcells. Similar methods can elucidate polypeptide markers by utilizingpolypeptide analytical techniques (e.g., PAGE, SDS-PAGE, procedurescomprising antibodies, and HPLC techniques known in the art).

Identification of Totipotent Cells

Totipotent cells can be identified by a number of tests. Examples ofthese tests include:

(1) identifying a marker specific for totipotent cells;

(2) performing one or more nuclear transfer cycles with a cell (asdescribed hereafter) and developing the resulting embryo into an animal.

Markers can be utilized to distinguish totipotent cells fromnon-totipotent cells. Markers can be selected from the group of lowmolecular weight markers, macromolecular markers, cell surface markers,and intracellular markers. Examples of markers that may be suitable foridentifying totipotent cells can be selected from the group consistingof alkaline phosphatase, cytokeratin, vimentin, laminin, and c-kit.These markers are well known to a person of ordinary skill in the artand these examples are not meant to be limiting.

Some of these markers have been tested for cultured bovine cells beingidentified for totipotency. As noted previously, totipotent,immortalized bovine cells of the invention generally do not appreciablystain for alkaline phosphatase. Therefore the cells of the invention areto be contrasted with pluripotent cells discussed in previouslyreferenced publications. It should be noted that some of the exemplarymarkers listed previously may not be specific for totipotent cells assome of these markers may exist in pluripotent cells as well as intotipotent cells. For example, although immortalized, totipotent bovinecells do not appreciably stain for alkaline phosphatase, immortalized,totipotent porcine cells may appreciably stain for alkaline phosphatase.The invention relates to any markers specific for totipotent cells thatare known to a person of ordinary skill in the art.

Markers for totipotency that are not clearly defined in the art can beelucidated by processes defined in the previous section, whichillustrates methods for elucidating immortalization cell markers.

A preferred test for determining totipotency of cells is determiningwhether cells give rise to totipotent embryos and eventually clonedanimals. This test represents a definitive test for cellulartotipotency. An example of such a test includes the following steps: (1)utilizing a potentially totipotent cell for nuclear transfer with anenucleated oocyte; (2) allowing the resulting cybrid to develop; (3)separating an embryo that developed from the cybrid into individualcells and subjecting one or more of the individual cells to a secondround of nuclear transfer; (4) allowing a resulting cybrid from step (3)to develop into an embryo; (5) implanting the embryo from step (2) or(4) into a uterine environment; and (6) allowing the embryo to develop.If the ensuing fetus develops past the first trimester of pregnancy thenthe cells initially used for nuclear transfer are most likely totipotentcells. If the cells utilized for nuclear transfer develop into a liveborn cloned animal then the cells are definitively totipotent. Examplesof the techniques utilized for this exemplary test (e.g., enucleation ofoocytes and nuclear transfer) are described completely in the art and inexemplary embodiments defined hereafter.

Hence, the materials and methods provided herein are the first tofeature immortalized, totipotent cells for cloning a bovine animal. Asdescribed above these materials and methods can be applied to otherungulates due to the high degree of nuclear DNA sequence homology amongungulates. Using the tests for identifying immortalized, totipotentcells, the methods and materials described herein can be modified by aperson of ordinary skill in the art to produce immortalized, totipotentcells from any type of precursor cell. Hence, the invention covers anyof the materials and methods described herein as well as modificationsto these methods for generating immortalized, totipotent cells, since aperson of ordinary skill in the art can readily produce immortalized,totipotent cells by utilizing the materials and methods described hereinin conjunction with methods for identifying immortalized, totipotentcells.

II. Transgenic Immortalized and Totipotent Cells

Materials and methods readily available to a person of ordinary skill inthe art can be utilized to convert immortalized, totipotent cells of theinvention into transgenic immortalized, totipotent cells. Once thenuclear DNA is modified in the immortalized, totipotent cells of theinvention, embryos and animals arising from these cells can alsocomprise the modified nuclear DNA. Hence, materials and methods readilyavailable to a person of ordinary skill in the art can be applied to theimmortalized, totipotent cells of the invention to produce transgenicanimals and chimeric animals. See, e.g., EPO 264 166, entitled“Transgenic Animals Secreting Desired Proteins Into Milk”; WO 94/19935,entitled “Isolation of Components of Interest From Milk”; WO 93/22432,entitled “Method for Identifying Transgenic Pre-implantation Embryos”;and WO 95/17085, entitled “Transgenic Production of Antibodies in Milk,”all of which are incorporated by reference herein in their entiretyincluding all figures, drawings and tables.

Methods for generating transgenic cells typically include the steps of(1) assembling a suitable DNA construct useful for inserting a specificDNA sequence into the nuclear genome of a cell; (2) transfecting the DNAconstruct into the cells; (3) allowing random insertion and/orhomologous recombination to occur. The modification resulting from thisprocess may be the insertion of a suitable DNA construct(s) into thetarget genome; deletion of DNA from the target genome; and/or mutationof the target genome.

DNA constructs can comprise a gene of interest as well as a variety ofelements including regulatory promoters, insulators, enhancers, andrepressors as well as elements for ribosomal binding to the RNAtranscribed from the DNA construct. DNA constructs can also encoderibozymes and anti-sense DNA and/or RNA, identified previously herein.These examples are well known to a person of ordinary skill in the artand are not meant to be limiting.

Due to the effective recombinant DNA techniques available in conjunctionwith DNA sequences for regulatory elements and genes readily availablein data bases and the commercial sector, a person of ordinary skill inthe art can readily generate a DNA construct appropriate forestablishing transgenic cells using the materials and methods describedherein.

Transfection techniques are well known to a person of ordinary skill inthe art and materials and methods for carrying out transfection of DNAconstructs into cells are commercially available. Materials typicallyused to transfect cells with DNA constructs are lipophilic compounds,such as Lipofectin™ for example. Particular lipophilic compounds can beinduced to form liposomes for mediating transfection of the DNAconstruct into the cells.

Target sequences from the DNA construct can be inserted into specificregions of the nuclear genome by rational design of the DNA construct.These design techniques and methods are well known to a person ofordinary skill in the art. See, U.S. Pat. No. 5,633,067, “Method ofProducing a Transgenic Bovine or Transgenic Bovine Embryo,” DeBoer etal., issued May 27, 1997; U.S. Pat. No. 5,612,205, “HomologousRecombination in Mammalian Cells,” Kay et al., issued Mar. 18, 1997; andPCT publication WO 93/22432, “Method for Identifying TransgenicPre-Implantation Embryos,” both of which are incorporated by referenceherein in their entirety, including all figures, drawings, and tables.Once the desired DNA sequence is inserted into the nuclear genome, thelocation of the insertion region as well as the frequency with which thedesired DNA sequence has inserted into the nuclear genome can beidentified by methods well known to those skilled in the art.

Once the transgene is inserted into the nuclear genome of theimmortalized, totipotent cell, that cell can be used as a nuclear donorfor cloning a transgenic animal. A description of the embodimentsrelated to transgenic animals are described in more detail hereafter.

A. Diseases and Parasites

Desired DNA sequences can be inserted into the (nuclear cellular) genometo enhance the resistance of a cloned transgenic animal to particularparasites and diseases. Examples of parasites include worms, flies,ticks, and fleas. Examples of infectious agents include bacteria, fungi,and viruses. Examples of diseases include Johne's, BVD, tuberculosis,foot and mouth, BLV, BSE and brucellosis. These examples are notlimiting and the invention relates to any disease or parasite orinfectious agent known in the art. See, e.g., Hagan & Bruners InfectiousDiesases of Domestic Animals (7th edition), Gillespie & Timoney,copyright 1981, Cornell University Press, Ithaca N.Y.

A transgene can confer resistance to a particular parasite or disease bycompletely abrogating or partially alleviating the symptoms of thedisease or parasitic condition, or by producing a protein which controlsthe parasite or disease.

B. Elements of DNA Constructs and Production of DNA Constructs

A wide variety of transcriptional and translational regulatory sequencesmay be employed, depending upon the nature of the host. Thetranscriptional and translational regulatory signals may be derived fromviral sources, such as adenovirus, bovine papilloma virus,cytomegalovirus, simian virus, or the like, whereas the regulatorysignals are associated with a particular gene sequence possessingpotential for high levels of expression. Alternatively, promoters frommammalian expression products, such as actin, casein, alpha-lactalbumin,uroplakin, collagen, myosin, and the like, may be employed.Transcriptional regulatory signals may be selected which allow forrepression or activation, so that expression of the gene product can bemodulated. Of interest are regulatory signals which can be repressed orinitiated by external factors such as chemicals or drugs. Other examplesof regulatory elements are described herein.

C. Examples of Preferred Recombinant Products

A variety of proteins and polypeptides can be encoded by a gene harboredwithin a DNA construct suitable for creating transgenic cells. Thoseproteins or polypeptides include hormones, growth factors, enzymes,clotting factors, apolipoproteins, receptors, drugs, pharmaceuticals,bioceuticals, nutraceuticals, oncogenes, tumor antigens, tumorsuppressors, cytokines, viral antigens, parasitic antigens, bacterialantigens and chemically synthesized polymers and polymers biosynthesizedand/or modified by chemical, cellular and/or enzymatic processes.Specific examples of these compounds include proinsulin, insulin, growthhormone, androgen receptors, insulin-like growth factor I, insulin-likegrowth factor II, insulin growth factor binding proteins, epidermalgrowth factor, TGF-α, TGF-β, dermal growth factor (PDGF), angiogenesisfactors (acidic fibroblast growth factor, basic fibroblast growth factorand angiogenin), matrix proteins (Type IV collagen, Type VII collagen,laminin), oncogenes (ras,fos, myc, erb, src, sis, jun), E6 or E7transforming sequence, p53 protein, cytokine receptor, IL-1, IL-6, IL-8,IL-2, α, β, or γIFN, GMCSF, GCSF, viral capsid protein, and proteinsfrom viral, bacterial and parasitic organisms. Other specific proteinsor polypeptides which can be expressed include: phenylalaninehydroxylase, α-1-antitrypsin, cholesterol-7α-hydroxylase, truncatedapolipoprotein B, lipoprotein lipase, apolipoprotein E, apolipoproteinA1, LDL receptor, scavenger receptor for oxidized lipoproteins,molecular variants of each, VEGF, and combinations thereof. Otherexamples are clotting factors, apolipoproteins, drugs, tumor antigens,viral antigens, parasitic antigens, monoclonal antibodies, and bacterialantigens. One skilled in the art readily appreciates that these proteinsbelong to a wide variety of classes of proteins, and that other proteinswithin these classes can also be used. These are only examples and arenot meant to be limiting in any way.

It should also be noted that the genetic material which is incorporatedinto the cells from DNA constructs includes (1) nucleic acid sequencesnot normally found in the cells; (2) nucleic acid molecules which arenormally found in the cells but not expressed at physiologicalsignificant levels; (3) nucleic acid sequences normally found in thecells and normally expressed at physiological desired levels; (4) othernucleic acid sequences which can be modified for expression in cells;and (5) any combination of the above.

In addition, DNA constructs may become incorporated into the nuclear DNAof cells, where the incorporated DNA can be transcribed into ribonucleicacid molecules that can cleave other RNA molecules at specific regions.Ribonucleic acid molecules which can cleave RNA molecules are referredto in the art as ribozymes, which are RNA molecules themselves.Ribozymes can bind to discrete regions on a RNA molecule, and thenspecifically cleave a region within that binding region or adjacent tothe binding region. Ribozyme techniques can thereby decrease the amountof polypeptide translated from formerly intact message RNA molecules.

Furthermore, DNA constructs can be incorporated into the nuclearcomplement of cells and when transcribed produce RNA that can bind toboth specific RNA or DNA sequences. The nucleic acid sequences areutilized in anti-sense techniques, which bind to the message (mRNA) andblock the translation of these messages. Anti-sense techniques canthereby block or partially block the synthesis of particularpolypeptides in cells.

III. Nuclear Transfer

Nuclear transfer (NT) techniques using non-immortalized andnon-totipotent cells are well known to a person of ordinary skill in theart. See, U.S. Pat. No. 4,994,384 (Prather et al.) and U.S. Pat. No.5,057,420 (Massey et al.). All of the advantages inherent to using theimmortalized, totipotent cells as described above are also advantagesfor NT techniques, specifically the fact that the immortalized,totipotent cells are a nearly unlimited source of nuclear donors andthat these cells increase the efficiency of NT. Exemplary embodimentsdefine a two-cycle NT technique that provides for efficient productionof totipotent bovine embryos. This technique can be applied to anymammal, preferably ungulates.

A. Nuclear Donors

Immortalized, totipotent cells of the invention can be used as nucleardonors in a NT process for generating a cloned embryo. As describedabove, the immortalized, totipotent cells can be generated from nearlyany type of cell. For NT techniques, a donor cell may be separated froma growing cell mass or isolated from a cell line. The entire cell may beplaced in the perivitelline space of a recipient oocyte or may bedirectly injected into the recipient oocyte by aspirating the nucleardonor into a needle, placing the needle into the recipient oocyte,releasing the nuclear donor and removing the needle withoutsignificantly disrupting the plasma membrane of the oocyte.Alternatively, a nucleus (karyoplast) may be isolated from a nucleardonor and placed into the perivitelline space of or injected directlyinto the recipient oocyte, for example.

B. Recipient Oocytes

A recipient oocyte is typically an oocyte with a portion of its ooplasmremoved, where the removed ooplasm comprises the oocyte nucleus.Enucleation techniques are well known to a person of ordinary skill inthe art. See e.g., U.S. Pat. Nos. 4,994,384 and 5,057,420.

Oocytes can be isolated from either oviducts and/or ovaries of liveanimals by oviductal recovery procedures or transvaginal oocyte recoveryprocedures well known in the art and described herein. Furthermore,oocytes can be isolated from deceased animals. For example, ovaries canbe obtained from abattoirs and the oocytes aspirated from these ovaries.The oocytes can also be isolated from the ovaries of a recentlysacrificed animal or when the ovary has been frozen and/or thawed.

Oocytes can be matured in a variety of media well known to a person ofordinary skill in the art. One example of such a medium suitable formaturing oocytes is depicted in an exemplary embodiment describedhereafter. Oocytes can be successfully matured in this type of mediumwithin an environment comprising 5% CO₂ at 39° C. Oocytes may becryopreserved and then thawed before placing the oocytes in maturationmedium. Cryopreservation procedures for cells and embryos are well knownin the art as discussed herein.

The nuclear donor may be incorporated into either a young or an agedoocyte. The age of the oocyte can be determined by the time that haselapsed since the oocyte was placed in maturation medium and the time itwas activated. A young oocyte can be defined as an oocyte that iscultured in vitro less than 28 hours before activation. An aged oocyteis defined as an oocyte that is cultured in vitro for more than 28 hoursbefore activation.

The age of the oocytes can be functionally identified by the appearanceof their ooplasm. For example, because certain cellular materials havenot yet dispersed within the ooplasm of a young oocyte, young oocyteshave a pycnotic appearance. Aged oocytes, in comparison, arecharacterized by a more homogeneous cytoplasm. A publication discussingthe use of aged oocytes for NT is WO 97/07662, entitled “InactivatedOocytes as Cytoplast Recipients for Nuclear Transfer.”

The nuclear donor cell and the recipient oocyte can arise from the samespecie or different species. For example, a bovine immortalized,totipotent cell can be inserted into a bovine enucleated oocyte.Alternatively, an immortalized, totipotent cell derived from a bison canbe inserted into a bovine enucleated oocyte. Any nuclear donor/recipientoocyte combinations are envisioned by the invention. Preferably thenuclear donor and recipient oocyte arise from one specie or differentspecies of ungulates. Cross-species NT techniques can be utilized toproduce cloned animals that are endangered.

The oocytes can be activated by electrical and/or non-electrical meansbefore, during, and/or after fusion of the nuclear donor and recipientoocyte. For example, the oocyte can be placed in a medium containing oneor more components suitable for non-electrical activation prior tofusion. Alternatively, a fused cybrid can be placed in a mediumcontaining one or more components suitable for non-electricalactivation. The activation process is discussed in greater detailhereafter.

C. Injection/Fusion

A nuclear donor can be translocated into an oocyte using a variety ofmaterials and methods that are well known to a person of ordinary skillin the art. In one example, a nuclear donor may be directly injectedinto a recipient oocyte. This direct injection can be accomplished bygently pulling a nuclear donor into a needle, piercing a recipientoocyte with that needle, releasing the nuclear donor into the oocyte,and removing the needle from the oocyte without significantly disruptingits membrane. Appropriate needles can be fashioned from glass capillarytubes, as defined in the art and specifically by publicationsincorporated herein by reference.

In another example, at least a portion of plasma membrane from a nucleardonor and recipient oocyte can be fused together using techniques wellknown to a person of ordinary skill in the art. See, Willadsen, 1986,Nature 320:63-65, hereby incorporated herein by reference in itsentirety including all figures, tables, and drawings. Typically, lipidmembranes can be fused together by electrical and chemical means, asdefined previously and in other references incorporated by referenceherein.

Other examples of non-electrical means of cell fusion involve incubatingcybrids in solutions comprising polyethylene glycol (PEG), and/or Sendaivirus. Various molecular weights of PEG can be utilized for cell fusion.

Although the efficiency of NT as a process is sensitive to minormodifications, other variables for fusion can be determined withoutundue experimentation. For example, modifications to cell fusiontechniques can be monitored for their efficiency by viewing the degreeof cell fusion under a microscope. The resulting embryo can then becloned and identified as a totipotent embryo by the same methods asthose previously described herein for identifying immortalized,totipotent cells, which can include tests for selectable markers and/ortests for developing an animal.

D. Activation

Methods of activating oocytes and cybrids are known to those of ordinaryskill in the art. See, U.S. Pat. No. 5,496,720, “Parthenogenic OocyteActivation,” Susko-Parrish et al., issued on Mar. 5, 1996, herebyincorporated by reference herein in its entirety including all figures,tables, and drawings.

Both electrical and non-electrical means can be used for activating thecybrids. Although use of a non-electrical means for activation is notalways necessary, non-electrical activation can enhance thedevelopmental potential of cybrids, particularly when young oocytes areutilized as recipients.

Examples of electrical techniques for activating cells are well known inthe art. See, U.S. Pat. Nos. 4,994,384 and 5,057,420. Non-electricalmeans for activating cells can include any method known in the art thatincreases the probability of cell division. Examples of non-electricalmeans for activating a nuclear donor and/or recipient can beaccomplished by introducing cells to ethanol; inositol trisphosphate(IP₃); Ca⁺⁺ ionophore and protein kinase inhibitors such as6-dimethylaminopurine; temperature change; protein synthesis inhibitors(e.g., cyclohexamide); phorbol esters such as phorbol 12-myristate13-acetate (PMA); mechanical techniques, thapsigargin, and spermfactors. Sperm factors can include any component of a sperm. Othernon-electrical methods for activation include subjecting the cell orcells to cold shock and/or mechanical stress.

Examples of preferred protein kinase inhibitors are protein kinase A, G,and C inhibitors such as 6-dimethylaminopurine (DMAP), staurosporin,2-aminopurine, sphingosine. Potentially, tyrosine kinase inhibitors mayalso be utilized to activate cells.

Although the NT process is sensitive to minor modifications, othervariables for activation can be determined without undueexperimentation. Other activation materials and methods can beidentified by modifying the specified conditions defined in theexemplary protocols described hereafter and in U.S. Pat. No. 5,496,720.

The result of any modifications upon efficiency and totipotency of theactivated embryo can be identified by the methods described previouslyin the section entitled “Identification of Immortalized and TotipotentCells.” Methods for identifying totipotent embryos can include one ormore tests, such as (a) identifying specific markers for totipotentcells in embryos, and (b) by determining whether the embryos aretotipotent by allowing them to develop into an animal. Therefore, theinvention relates to any modifications to the activation proceduresdescribed herein even though these modifications may not be explicitlystated herein.

F. Manipulation of Embryos Resulting from Nuclear Transfer

An embryo resulting from a NT can be manipulated in a variety ofmanners. The invention relates to cloned embryos that arise from atleast one NT.

Exemplary embodiments of the invention demonstrate that two or more NTprocedures may enhance the efficiency for the production of totipotentembryos. The exemplary embodiments indicate that incorporating two ormore NT procedures into methods for producing cloned totipotent embryosmay enhance placental development. In addition, increasing the number ofNT cycles involved in a process for producing totipotent embryos mayrepresent a necessary factor for converting non-totipotent cells intototipotent cells. The effect of incorporating two or more NT cycles onthe totipotency of resulting embryos is a surprising result, which wasnot previously identified or explored in the art.

Incorporating two or more NT cycles into methods for cloned totipotentembryos can provide another advantage. Incorporation of multiple NTprocedures into methods for creating cloned totipotent embryos providesa method for multiplying the number of cloned totipotent embryos.

When multiple NT procedures are utilized for the formation of a clonedtotipotent embryo, young or aged oocytes can be utilized as recipientsin the first, second or subsequent NT procedures. For example, if afirst NT and then a second NT are performed, the first NT can utilize ayoung enucleated oocyte as a recipient and the second NT may utilize anaged enucleated oocyte as a recipient. Alternatively, the first NT mayutilize an aged enucleated oocyte as a recipient and the second NT mayutilize a young enucleated oocyte as a recipient for the same two-cyclemodel for NT. In addition, both NT cycles may utilize young enucleatedoocytes as recipients or both NT cycles may utilize aged enucleatedoocytes as recipients in the two-cycle NT example.

For NT techniques that incorporate two or more NT cycles, one or more ofthe NT cycles may be preceded, followed, and/or carried outsimultaneously with an activation step. As defined previously herein, anactivation step may be accomplished by electrical and/or non-electricalmeans as defined herein. Exemplified embodiments described hereafterdescribe NT techniques that incorporate an activation step after one ofthe NT cycles. However, activation steps may also be carried out inconjunction with NT cycles (e.g., simultaneously with the NT cycle)and/or activation steps may be carried out prior to a NT cycle.

A preferred embodiment of the invention, for example, relates to a firstNT utilizing a young enucleated oocyte as a recipient followed byactivation. This, in turn, is followed by a second NT utilizing an agedenucleated oocyte as a recipient. This second NT procedure is notfollowed by activation. This example is not meant to be limiting and theinvention relates to any number of NT cycles that are optionallypreceded by, followed by, simultaneously carried out with an activationprocedure.

NT techniques may utilize virtually any cell as a nuclear donor. Forexample, in a preferred embodiment, a first NT may utilize animmortalized, totipotent cell of the invention as a nuclear donor and asecond NT may utilize an embryonic cell as a nuclear donor. The secondNT cycle in this example may utilize a blastomere (a cell isolated froman embryo), a cell isolated from a fetus (e.g., a primordial germ cell)as a nuclear donor, a cell isolated from a cell line, or a synchronizedcell (described herein). The invention pertains in part to utilizingnearly any type of cell as a nuclear donor in any NT. The effect ofusing different nuclear donors on the overall efficiency for producingcloned totipotent embryos can be tested by practicing the tests fortotipotency described in the preceding section entitled “Identificationof Immortalized and Totipotent Cells.”

The cloned totipotent embryos resulting from NTs can be (1)disaggregated or (2) allowed to develop further.

If the embryos are disaggregated, these embryonic derived cells can beutilized to establish cultured cells. Any type of embryonic cell can beutilized to produce cultured cells. These cultured cells are sometimesreferred to as embryonic stem cells or embryonic stem-like cells in thescientific literature. The embryonic stem cells can be derived fromearly embryos, morulae, and blastocyst stage embryos. Multiple methodsare known to a person of ordinary skill in the art for producingcultured embryonic cells. These methods are enumerated in specificreferences previously incorporated by reference herein.

If the embryos are allowed to develop in utero, cells isolated from thedeveloping fetus can be utilized to produce cultured cells. In preferredembodiments, primordial germ cells are isolated from the genital ridgeof 28 to 75 day old developing cell masses for the establishment of celllines. These cultured cells are sometimes referred to as embryonic germcells (EG). These cultured cells can be generated using methods wellknown to a person of ordinary skill in the art. The methods areenumerated in references previously incorporated by reference herein.

The cloned totipotent embryos resulting from NT can also be manipulatedby cryopreserving and/or thawing the embryos. See, U.S. Pat. No.5,160,312, entitled “Cryopreservation Process for Direct Transfer ofEmbryos,” Voelkel, and issued on Nov. 3, 1992; and U.S. Pat. No.4,227,381, entitled “Wind Tunnel Freezer,” Sullivan et al., issued onOct. 14, 1980, all of which are hereby incorporated by reference hereinin their entireties including all tables, figures, and drawings. Otherembryo manipulation methods include culturing, performing embryotransfer, dissociating for NT, dissociating for establishing cell linesfor use in NT, splitting, aggregating, sexing, and biopsying the embryosresulting from NT, which are described hereafter. The exemplarymanipulation procedures are not meant to be limiting and the inventionrelates to any embryo manipulation procedure known to a person ofordinary skill in the art.

IV. Development of Cloned Embryos

A. Totipotency

Totipotent embryos can be identified by the methods described in thesection “Identification of Immortalized and Totipotent Cells.”Individual cells can be isolated and subjected to these similar tests.The tests relate to similar markers for identifying totipotent cells, aswell as a test for determining totipotency by allowing an embryo todevelop until it passes the second trimester of gestation, orpreferably, gives rise to a live born animal. Methods for identifyingother markers for totipotency are also described in that section.

B. Culture of Embryos in vitro

Methods for culturing embryos in vitro are well known to those skilledin the art. See, U.S. Pat. No. 5,213,979, entitled “In vitro Culture ofBovine Embryos,” First et al., issued on May 25, 1993, and U.S. Pat. No.5,096,822, entitled “Bovine Embryo Medium,” Rosenkrans, Jr. et al.,issued on Mar. 17, 1992, both of which are incorporated by referenceherein in its entirety, including all figures, tables, and drawings. Inaddition, exemplary embodiments for media suitable for culturing clonedembryos in vitro are described hereafter. Feeder cell layers may or maynot be utilized for culturing cloned embryos in vitro. Feeder cells aredescribed previously and in exemplary embodiments hereafter.

The present invention is superior to existing materials and methods forcloning organisms, because embodiments of the invention allow forculturing all cells and embryos in vitro prior to implantation. Forexample, cloning methods described for cloning ovine organisms requirean in vivo development step in the oviducts of an ovine host animalbefore the embryos are implanted in a suitable host. Because embodimentsof the present invention do not require in vivo development steps priorto implantation into the uterus, the materials and methods of thepresent invention represent an inventive step over cloning methodspreviously described by others.

C. Development of Embryos in utero

Cloned embryos can be cultured in an artificial or natural uterineenvironment after NT procedures. Examples of artificial developmentenvironments are being developed and some are known to those skilled inthe art. Components of the artificial environment can be modified withlittle experimentation, for example, by modifying one component andmonitoring the growth rate of the embryo.

Methods for implanting embryos into the uterus of an animal are alsowell known in the art. Preferably, the developmental stage of theembryo(s) is correlated with the estrus cycle of the animal.

Embryos from one specie can be placed into the uterine environment of ananimal from another specie. For example it has been shown in the artthat bovine embryos can develop in the oviducts of sheep. Stice &Keefer, 1993, “Multiple generational bovine embryo cloning,” Biology ofReproduction 48: 715-719. The invention relates to any combination ofungulate embryo in any other ungulate uterine environment. Thecross-species relationship between embryo and uterus can allow forefficient production of cloned animals of an endangered species. Forexample, a bison embryo can develop in the uterus of a domestic bovine.In another example, a big-horn sheep embryo can develop in the uterus ofa large domesticated sheep.

Once the embryo is placed in the uterus of an animal, the embryo candevelop to term. Alternatively, the embryo can be allowed to develop inthe uterus and then can be removed at a chosen time. Surgical methodsare well known in the art for removing fetuses from uteri before theyare born.

V. Cloned Animals

A. Bovine Cloned Animals

As described previously herein, the invention provides the advantages ofbeing able to assess the phenotype of an animal before cloning. This isan advantage of the invention since previous reports have only allowedthe cloning of bovine animals from blastomeres, a method that does notallow for phenotype assessment.

Multiple products can be isolated from a cloned animal. For example,semen can be collected from an animal, such as a bovine bull. Semen canbe cryopreserved as well as separated sperm into sex-specific fractions.See, U.S. Pat. Nos. 5,439,362, 5,346,990, and 5,021,244, entitled“Sex-associated Membrane Proteins and Methods for Increasing theProbability that Offspring Will be of a Desired Sex,” Spaulding, andissued on Aug. 8, 1995, Sep. 13, 1994, and Jun. 4, 1991, respectively,all of which are hereby incorporated by reference herein in theirentireties including all figures, drawings, and tables. Methods ofcollecting semen are well known to a person of ordinary skill in theart. Physiology of Reproduction and Artificial Insemination of Cattle(2nd. edition), Salisbury et al., copyright 1961, 1978, W.H. Freeman &Co., San Francisco.

The invention relates in part to any products collected from a clonedanimal, preferably a cloned bovine animal. The products can be any bodyfluids or organs isolated from the animal, or any products isolated fromthe fluids or organs. In preferred embodiments, products such as milkand meat may be collected from cloned animals, preferably cloned bovineanimals. In another embodiment, the invention relates to determining thephenotype of a bovine steer, which is a neutered animal, and thencloning this animal such that the cloned animals are reproductivelyfunctional and can be used to produce semen. Other preferred embodimentsof the invention relate to such products as xenograft materials, sperm,embryos, oocytes, any type of cells, and offspring harvested from clonedanimals of the invention, preferably cloned bovine animals.

Xenograft materials, which are described previously herein, can relateto any cellular material extracted from one organism and placed intoanother organism. Medical procedures for extracting the cellularmaterial from one organism and grafting it into another organism arewell known to a person of ordinary skill in the art. Examples ofpreferable xenograft cellular materials can be selected from the groupconsisting of liver, lung, heart, nerve, gallbladder, and pancreascellular material.

B. Non-Bovine Cloned Animals

Due to the high DNA sequence homology between bovine animals and otherungulates, the materials and methods of the invention can be utilized toclone other ungulates. The materials and methods of the invention arethe most efficient means for cloning a mammal as known in the state ofthe art.

In preferred embodiments the materials and methods of the invention canbe utilized to clone endangered species, such as bison. In addition, thematerials and methods of the invention can be utilized to clonecommercially relevant ungulates, such as pigs. Due to the methods forreprogramming primary cells isolated from an animal into immortalized,totipotent cells, the more closely related the animal species is tocattle, the higher probability that the cloning methods of the inventionwill have greater success. Exemplary embodiments are described hereafterfor cloning non-bovine animals.

C. Cloned Animals with Modified Nuclear DNA

As discussed in a previous section, transgenic animals can be generatedfrom the methods of the invention by using transgenic techniques wellknown to those of ordinary skill in the art. Preferably, clonedtransgenic bovine animals are produced from these methods. These clonedtransgenic animals can be engineered such that they are resistant orpartially resistant to diseases and parasites endemic to such animals.Examples of these diseases and parasites are outlined in a precedingsection.

Moreover, the cloned transgenic animals can be engineered such that theyproduce a recombinant product. Examples of recombinant products areoutlined in a preceding section. The expression of these products can bedirected to particular cells or regions within the cloned transgenicanimals by selectively engineering a suitable promoter element and otherregulatory elements to achieve this end.

For example, human recombinant products can be expressed in the urine ofcattle by operably linking a uroplakin promoter to the DNA sequenceencoding a recombinant product. Alternatively, examples are well knownto a person of ordinary skill in the art for selectively expressinghuman recombinant products in the milk of a bovine animal.

Once the recombinant product or products have been expressed in aparticular tissue or fluid of the cloned transgenic animal, the suitabletissue or fluid can be collected using methods well known in the art.Recombinant products can be purified from that fluid or tissue by usingstandard purification techniques well known to a person of ordinaryskill in the art.

EXAMPLES

The examples below are non-limiting and are merely representative ofvarious aspects and features of the present invention.

Example 1 Feeder Layer Preparation

A feeder cell layer was prepared from mouse fetuses that were from 10 to20 days gestation. The head, liver, heart and alimentary tract wereremoved and the remaining tissue washed and incubated at 37° C. in0.025% trypsin-0.02%, EDTA (Difco, Cat # 0153-61-1). Loose cells werecultured in tissue culture dishes containing MEM-alpha supplemented withpenicillin, streptomycin, 10% fetal calf serum and 0.1 mM2-mercaptoethanol. The feeder cell cultures were established over a twoto three week period at 37.4° C., 3.5% CO₂ and humidified air. Beforebeing used as feeder cells, mouse fibroblasts were pre-treated withmitomycin C (Calbiochem, Cat # 47589) at a final concentration of 10μg/ml for 3 hours and washed 5 times with PBS before pre-equilibratedgrowth media was added.

Feeder cells can be established from bovine fetuses from 30 to 70 daysusing the same procedure. Bovine fetal cells may be optionally treatedwith mitomycin C.

Example 2 Establishing Cultured Cells from Non-Embryonic Tissue

One advantage provided by the materials and methods defined herein isthe ability to create an immortalized and totipotent cell from virtuallyany type of precursor cell. These precursor cells can be embryoniccells, cultured embryonic cells, primordial germ cells, fetal cells, andcells isolated from the tissues of adult animals, for example. Cellsisolated from the kidney and ear of an adult grown bovine have beenutilized as precursor cells for the generation of immortalized,totipotent cells.

After cells are isolated from their respective tissues, the cells can besubjected to the materials and methods defined in Example 3.

A first step towards generating immortalized, totipotent cells fromtissues of grown animals includes a primary culture of isolated cells. Aprotocol for culturing cells isolated from the tissues of grown animalsis provided hereafter. Although the illustrative protocol relates to earpunch samples, this protocol can apply to cells isolated from any typeof tissue.

The following steps are preferably performed utilizing sterileprocedures:

1) Wash each ear sample twice with 2 mL of trypsin/EDTA solution in twoseparate 35 mm Petri dishes. Process each ear sample separately. Mincethe ear sample with sterile scissors and scalpel in a 35 mm Petri dishcontaining 2 mL of trypsin/EDTA solution. The minced pieces arepreferably less than 1 mm in diameter.

2) Incubate minced ear pieces in the trypsin/EDTA solution for 40-50min. in a 37° C. incubator with occasional swirling. The trypsin/EDTAsolution is described in more detail hereafter. The dish may be wrappedwith a stretchable material, such as Parafilm®, to reduce CO₂accumulation.

3) Transfer digested ear pieces to a 15 mL sterile tube. Wash the dishfrom which the digested ear pieces were recovered with 2 mL of thetrypsin/EDTA solution and transfer this wash solution to the steriletube.

4) Vortex the tube at high speed for 2 min.

5) Add 5 mL of media (defined below) to inactivate the trypsin.

6) Centrifuge the 15 mL tube at 280×g for 10 minutes.

7) Decant the supernatant and re-suspend the cell pellet in residualsolution by gently taping the side of the tube.

8) Add 2 mL of media to the tube and then centrifuge as described instep (6).

9) Decant the supernatant, re-suspend the pellet as described in step(7), then add 2 mL of media.

10) Keep 2-3 pieces of the ear for DNA analysis and store at −20° C. ina 15 mL tube.

11) Transfer resuspended cells into a 35 mm Nunc culture dish andincubate at 37° C. in a humidified 5% CO₂/95% air atmosphere.

12) Change media every 2 days.

Trypsin/EDTA Solution

0.025% trypsin (w/v) (Bacto trypsin, Difco #cat 0153-61-1)

0.02% EDTA (Sigma) (w/v)

Add the trypsin and EDTA to Ca²⁺-free and Mg²⁺-free Dulbecco'sphosphate-buffered saline (PBS) (Gibco cat# 450-1600EA) and sterilize byfiltration through a 0.2 μm filter.

Media

Combine Alpha minimum essential medium (MEM) (Biowhittaker) with 10%fetal bovine serum (Hyclone), 4 mM L-glutamine, 100 U/mL penicillin, 100μg/ML streptomycin, 0.25 μg/mL amphotercin B (Fungizone).

This protocol has been also successfully utilized to establish culturesof kidney and liver cells isolated from grown bovine animals. Asdiscussed above, the protocol can be utilized to create cell culturesfrom any type of cell isolated from a grown animal, for any species orfamily of animals.

Example 3 Reprogramming and Establishment of Immortalized and TotipotentCells from Precursor Cells

The reprogramming procedures described hereafter can utilize any celltype of cells as precursor cells for the generation of immortalized,totipotent cells. As an example, the cell cultures described previouslycan be utilized as precursor cells for the reprogramming proceduresdescribed below. As another example, the following procedure describesone embodiment of the invention, where primordial germ cells wereutilized as precursor cells for the generation of immortalized,totipotent cells. An embodiment of the reprogramming process isillustrated in FIG. 2.

A bovine fetus approximately 40 days old was obtained from a pregnantanimal. The genital ridges were located at the caudo-ventral part of theabdominal cavity. Genital ridges were removed aseptically and washed inphosphate buffered saline (PBS) (Gibco, Cat # 14287-015) with 500 U/mLpenicillin/500 μg/ml streptomycin. The tissue was sliced into 1-1.5 mmpieces and placed into a solution containing pronase E (3 mg/ml; SigmaCat # P6911) in Tyrodes Lactate (TL) HEPES (Biowhittaker, Cat # 04-616F)for 30-45 minutes at 35-37° C. The proteolytic action of pronase Edisaggregated the slices of genital ridges to a cell suspension. PronaseE was removed by dilution and centrifugation in TL HEPES solution. Afterthis step, the cell suspension was frozen and stored at −196° C.

A thawed cell suspension (final concentration 100,000 cells/ml) wasplaced into a 35 mm Petri dish containing a murine primary embryonicfibroblast feeder layer. The culture media used was MEM alpha(Biowhittaker, Cat # 12-169F) supplemented with 0.1 mM 2-mercaptoethanol(Gibco, Cat # 21985-023), 4 mM glutamine, 100 ng/ml human recombinantleukemia inhibitory factor (hrLIF; R&D System, Cat # 250-L), 100 ng/mlbovine basic fibroblast growth factor (bFGF; R&D System, Cat # 133-FB)and 10% fetal calf serum (FCS, HyClone, Cat # A1111D) at 37.5° C. and3.5% CO₂. Exogenous steel factor (e.g., membrane associated steel factorand soluble steel factor) was not added to the culture media. After 24hours, and again at 48 hour intervals, supplemented culture media wasreplaced. After an initial culture of 6 days, concentrations of hrLIFand bFGF were lowered to 40 ng/ml, respectively. After nine days inculture, hrLIF and bFGF were removed from the medium entirely.

At the beginning of in vitro culture of genital ridge cells, simpleembryonic bodies were occasionally observed. These bodies eventuallydisappeared with subsequent passages. The rate of establishingimmortalized, totipotent cell lines from genital ridge cells was 100%and did not appear to be sex dependent. Table 1 contains data fromestablishment of seven immortalized, totipotent cell lines. Establishedimmortalized, totipotent cell lines were maintained in MEM-alphasupplemented with 10% FCS which was replaced every second or third day.High density population cells were passaged every week at a dilutionratio of 1:4 to 1:8. Cells were passaged by incubating with 0.025%trypsin-0.02% EDTA mixture and preparing new cultures in fresh growthmedium. The growth promoting capacity of MEM-alpha media forimmortalized, totipotent cells was enhanced by addinginsulin-transferrin, sodium selenite supplement, diluted to 1:100 (SigmaCat # 11884). As a preventive measure against mycoplasma contamination,short term cultivation with tylosine tartrate (Sigma, Cat T3151) wascarried out. Before NT, cell lines were tested for presence ofmycoplasma by PCR performed with DNA primers specific for mycoplasmasequences (Stratagene, Cat 302007).

TABLE 1 Characterization of Established Bovine Immortalized andTotipotent Cell Lines Cell line Weight of fetus (gm) Days in culture Sexof Cell line EG 14.2 >400 male EG-1 20.2 >300 male EG-2 3.9 >30 femaleEG-3 4.8 >30 male EG-4 39.6 >100 female EG-5 3.9 >250 male EG-6 8.6 >30male

Example 4 Embryo Construction

The following embodiment of the invention describes materials andmethods utilized to produce totipotent embryos of the invention.Immortalized embryos of the invention can be produced by utilizingimmortalized and totipotent cells of the invention as nuclear donors inNT procedures. As described previously, multiple NT procedures can beutilized to create a totipotent embryo. The following two examplesdescribe a multiple NT procedure, which describes the use of two NTs.

Mycoplasma free immortalized, totipotent cells used in the NT procedure,were prepared by cutting out a group of immortalized, totipotent cellsfrom the feeder layer using a glass needle. The isolated immortalized,totipotent cells were then incubated in a TL HEPES solution containingfrom 1 to 3 mg/ml pronase E at approximately 32° C. for 1 to 4 hours,the amount of time which was needed in this example to disaggregate thecells. Once the cells were in a single cell suspension they were usedfor NT within a 2-3 hour period.

Oocytes aspirated from ovaries were matured overnight (16 hours) inmaturation medium. Medium 199 (Biowhittaker, Cat #12-119F) supplementedwith luteinizing hormone 10 IU/ml (LH; Sigma, Cat # L9773), 1 mg/mlestradiol (Sigma, Cat # E8875) and 10% FCS or estrus cow serum, wasused. Within 16 hours of maturation, the cumulus layer expanded and thefirst polar bodies were extruded.

In the first NT procedure, young oocytes were stripped of their cumuluscell layers and nuclear material stained with Hoechst 33342 5 mg/ml(Sigma, Cat # 2261) in TL HEPES solution supplemented with cytochalasinB (7 μg/ml, Sigma, Cat # C6762) for 15 min. Oocytes were then enucleatedin TL HEPES solution under mineral oil. A single immortalized,totipotent cell of optimal size (12 to 15 μm) was then inserted from acell suspension and injected into the perivitelline space of theenucleated oocyte. The immortalized, totipotent cell and oocytemembranes were then induced to fuse by electrofusion in a 500 μm chamberby application of an electrical pulse of 90V for 15 μs.

Cybrid activation was induced by a 4 min exposure to 5 μM calciumionophore A23187 (Sigma Cat. # C-7522) or ionomycin Ca-salt in HECM(hamster embryo culture medium) containing 1 mg/ml BSA followed by a1:1000 dilution in HECM containing 30 mg/ml BSA for 5 min. For HECMmedium, see, e.g., Seshagiri & Barister, 1989, “Phosphate is requiredfor inhibition of glucose of development of hamster eight-cell embryosin vitro,” Biol. Reprod. 40: 599-606. This step is followed byincubation in CR2 medium containing 1.9 mM 6-dimethylaminopurine (DMAP;Sigma product, Cat # D2629) for 4 hrs followed by a wash in HECM andthen cultured in CR2 media with BSA (3 mg/ml) under humidified air with5% CO₂ at 39° C. For CR2 medium, see, e.g., Rosenkrans & First, 1994,“Effect of free amino acids and vitamins on cleavage and developmentalrate of bovine zygotes in vitro,” J. Anim. Sci. 72: 434-437. Mitoticdivisions of the cybrid formed an embryo. Three days later the embryoswere transferred to CR2 media containing 10% FCS for the remainder oftheir in vitro culture.

Table 2 shows the effect of oocyte age on blastocyst development. Thedata was obtained utilizing blastomeres from in vitro produced embryosor immortalized, totipotent cells as donor nuclei in the NT procedure.Developmental potential was measured in young versus aged oocytes.

TABLE 2 Effect of Oocyte Timing for Different Cell Sources Immortalizedand oocyte age (hours) Totipotent Cells (n = 174) Blastomeres (n = 192)16-28 (n = 175) 17.9% blastocyst (n = 40) no development (n = 35) 28-48(n = 191) no development (n = 34) 17.3% blastocyst (n = 157)

The data presented in Table 2 shows that oocytes maintained in culturefor 16-28 h were more suitable recipients for immortalized, totipotentcells, while aged oocytes maintained in culture for 28-48 h were a moresuitable recipient for blastomeres derived from embryos. In addition,activation procedures differed between young and aged oocytes. Youngoocytes, when used in the NT procedure, appear to require chemicalactivation with ionomycin and DMAP from these studies. Aged oocytes, onthe other hand, appear to be easily activated by electrofusion accordingto these studies.

Example 5 Second Nuclear Transfer (Recloning)

Cells obtained from fetuses and embryos produced by the NT proceduresdescribed herein can be used in a second NT, or recloning, procedure.For example, a fetus can be harvested from a maternal host, the head,vicera, and genital ridge removed, and the remaining fetal cells used toestablish a cell line to provide nuclear donor material for a subsequentNT procedure. The following example describes obtaining and using ablastomere from an NT embryo as a nuclear donor in a recloningprocedure.

Embryos from the first generation NT at the morula stage weredisaggregated either by pronase E (1-3 mg/ml in TL HEPES) ormechanically after treatment with cytochalasin B. Single blastomereswere placed into the perivitelline space of enucleated aged oocytes(28-48 hours of incubation). Aged oocytes were produced by incubatingmatured “young” oocytes for an additional time in CR2 media with 3 mg/mlBSA in humidified air with 5% CO₂ at 39° C.

A blastomere from an embryo produced from an immortalized, totipotentcell was fused into the enucleated oocyte via electrofusion in a 500 μmchamber with an electrical pulse of 105V for 15 μs in an isotonicsorbitol solution (0.25 M) at 30° C. Aged oocytes were simultaneouslyactivated with a fusion pulse, not by chemical activation as with youngoocytes.

After blastomere-oocyte fusion, the cybrids from second generation NTwere cultured in CR2 media supplemented with BSA (3 mg/ml) underhumidified air with 5% CO₂ at 39° C. On the third day of culture,developing embryos were evaluated and cultured further until day sevenin CR2 media containing 10% FCS. Morphologically good to fair qualityembryos were non-surgically transferred into recipient females. Table 3shows the increased gestation length achieved by use of recloned (doubleNT) immortalized, totipotent cells.

TABLE 3 Development of Immortalized and Totipotent Cells Derived Fetusesafter Double NT No. of No. of pregnant embryos No. of recipientsrecipients after No. of transferred transferred into 140 days calvesExper #1 15 5 1 1 Exper #2 18 6 1 (two fetuses) 2

Example 6 Cloning Non-Bovine Ungulates

The specification provides for methods of cloning non-bovine ungulates.Examples of such ungulates can be selected from the group consisting ofbovids, ovids, cervids, suids, equids and camelids, such as bison,sheep, big-horn sheep, caribou, antelope, deer, goat, water buffalo,camel, and pig.

Immortalized, totipotent cell lines can be prepared from multiple typesof cells isolated from the non-bovine ungulate by using the methodsdescribed in previous examples relating to bovine animals, or by usingthe screening procedures for these methods as described in thespecification. Virtually any type of cell isolated from the non-bovineungulate can be utilized to establish an immortalized, totipotent cellline. For example, an ear-punch sample taken from a bison can becultured in vitro using a variety of cell culture media such asMEM-alpha medium.

Bison-derived primary cells can then be converted or reprogrammed intoimmortalized, totipotent bison cells by supplementing the cell culturemedium with hrLIF and bFGF as described in previous examples and in thespecification. Alternatively, the bison-derived primary cells can beconverted into immortalized, totipotent cells by supplementing thegrowth medium with other types of molecules identified by methods foridentifying such reprogramming molecules as described in thespecification. The reprogrammed bison-derived cells can then be testedfor totipotency by analyzing selected markers, such as alkalinephosphatase, laminin, and c-kit. In addition, the bison-derived cellscan be considered permanent if the number of cell divisions exceeds theHayflick limit and/or if the cells can grow to confluency after beingreplated under conditions where the cells are not in physical contactwith one another, for example.

Once totipotent, immortalized cells have been established as nucleardonors, proper enucleated oocytes can be prepared for NT. Oocytes fromthe same or different specie as the nuclear donor can be used for NT.For example, a bison-derived nuclear donor cell can be fused or directlyinjected into a bison-derived enucleated oocyte or an enucleated oocytefrom another specie, such as a bovine.

As described in the specification, the oocytes can be derived from anyungulate in a variety of ways, such as sacrificing an animal andretrieving oocytes from its oviducts, or spaying the animals by ovarianhysterectomy and isolating the oocytes from the oviducts or ovaries.Oocytes can also be obtained from live animals by utilizing such methodsas transvaginal oocyte recovery. The oocytes can then be enucleated byusing methods described herein as applied to sheep or cattle. Thesemethods can be easily applied to oocytes derived from other ungulates.

Nuclear transfer techniques can be performed after enucleated oocytesand nuclear donor cells are prepared. Young or aged oocytes can beutilized for the NT procedure, and the number of NTs can vary asdescribed in the specification. In addition the parameters that definethe fusion step for a NT may be varied as described herein. Anactivation step can be applied to one or more of the NT cycles. Forexample, the NT cycles defined in a previous exemplary embodiment can beapplied to the generation of cloned bison. The embryo resulting from theNT can be tested for totipotency by utilizing tests for one or moremarkers, such as alkaline phosphatase, cytokeratin, vimentin, laminin,and c-kit. In addition, the embryo can be tested for totipotency byimplanting it into the uterus of an animal and allowing development toterm.

Once a cloned totipotent embryo is produced from the methods describedabove for a non-bovine ungulate, the embryo can be further manipulated.Such manipulations include cryopreserving, thawing, culturing,disaggregating the embryo into single cells, and implanting the embryo.The embryo may be cultured in an artificial development environment (asdescribed previously) or may be placed in utero of a properlysynchronized female animal. An embryo derived from one specie may beplaced in a uterus of the same or different specie. For example, abison-derived embryo can be placed in the uterus of a bovine. The embryocan be allowed to develop until term, or may be retrieved from theuterine environment before birth.

Example 7 Multiple Pathways for Cloning Animals

FIG. 3 illustrates multiple embodiments of the invention. Animals can becloned from cells that are reprogrammed into totipotent and immortalizedcells.

Fibroblast cell cultures were prepared as defined above from ear punchesextracted from an adult bovine animal. However, the cell cultures couldbe established from any type of differentiated cell. Individual cellsisolated from these cultures were utilized as nuclear donors in anuclear transfer process, labeled as step 2 in FIG. 3. Although onenuclear transfer cycle was utilized to obtain embryos (labeled as step 3in FIG. 3), multiple nuclear transfer cycles could be applied to obtainthese embryos. Also optional is (1) the addition of a stimulus before orafter nuclear transfer, and (2) an activation step before or afternuclear transfer.

The embryo of step 3 in FIG. 3 was implanted into a recipient bovinefemale as described herein and a fetus (step 7) was isolated from thatfemale. Cells isolated from embryos of step 3 may be utilized toestablish embryonic stem cell cultures (step 4). In addition, theembryos of step 3 may be implanted into a female host and allowed todevelop into a cloned animal (step 5).

The steps labelled 8, 9, 10, 11, and 12 in FIG. 3 were performed toestablish totipotent and immortalized cells. The fetus of step 7 wasmanipulated in three manners. The manipulation in step 8 involved theisolation of genital ridge cells, specifically primordial germ cells,from the fetus of step 7. In step 9, the primordial germ cells wereplaced in co-culture with feeder cells. The feeder cells were eitherestablished from mouse fibroblast cells or from the rest of the fetusfrom which the primordial germ cells were extracted. Example 1 defines amethod for establishing feeder cells. The head region and body cavitycontents were removed from the fetus before the fetus was digested intoa consistency suitable for establishing feeder cells. However, the fetusmay be digested before the head region and contents of the body cavityare removed. In addition, feeder cells may be established from a fetusother than the fetus from which the primordial germ cells are isolated.

In step 10, a cell culture was established with a digested fetus fromwhich the primordial germ cells, head region, and body cavity contentswere removed. Step 11 illustrates that cell cultures may be establishedutilizing fetuses from which no cell types have been removed.

In step 12, cell cultures were either (1) subjected to a mechanicalstimulus, or (2) not subjected to a mechanical stimulus. When applied,the mechanical stimulus was effected by supplementing the culture mediumwith a receptor ligand cocktail comprising 100 ng/ml human recombinantleukemia inhibitory factor (hrLIF; R&D System, Cat # 250-L) and 100ng/ml bovine basic fibroblast growth factor (bFGF; R&D System, Cat#133-FB). After step 12, cells were isolated from the cell cultures andutilized as nuclear donors in nuclear transfer processes, which aredefined previously. Although one nuclear transfer cycle was utilized forstep 13, more than one nuclear transfer cycle could be utilized.

Embryos developed after the nuclear transfer process of step 13. Theembryos of step 14 may be implanted into a bovine recipient female anddevelop into a cloned bovine animal.

Cells isolated from any of the developing cell masses of steps 1, 3, 4,5, 7, 8, 9, 10, 11, 12, 13, and 16 in FIG. 3 may be transfected with aDNA construct to form transgenic cells suitable for cloning transgenicanimals. One embodiment for cloning transgenic animals is defined in thenext example.

Example 8 Cloning Transgenic Animals

Transgenic cells suitable for creating a cloned transgenic animal can beprepared from cells isolated from an adult animal. FIG. 4 illustratesprocesses that can be utilized to create such transgenic cells. Althoughtransgenic cells can be created from nearly any cell type by using theteachings of the invention, FIG. 4 illustrates procedures forestablishing transgenic embryonic stem cells and transgenic immortalizedand totipotent cells.

Fibroblast cell cultures can be established from ear punches extractedfrom a bovine animal as defined previously. Individual cells can beisolated from this cell culture and utilized as nuclear donors in anuclear transfer process. A single nuclear transfer cycle or multiplenuclear transfer cycles can be applied. Other optional steps are definedin the previous example.

Pre-blastocyst stage embryos and/or blastocyst stage embryos developedfrom the nuclear transfer process can be utilized to establish embryonicstem cells. Materials and methods for preparing embryonic stem cells aredescribed by Stice et al., 1996, Biology of Reproduction 54: 100-110,hereby incorporated by reference herein in its entirety, including allfigures, tables, and drawings. Immortalized and totipotent cells can beestablished according to the procedures defined in previous examples.

Cells can then be transfected with a DNA construct. Cells can betransfected at multiple steps, as indicated in FIG. 4. Materials andmethods for preparing transgenic cells are defined in publicationsreferenced previously. Immortalized and totipotent cells of theinvention were successfully transfected with a DNA construct comprising(a) a neomycin gene, which encodes a product that renders cellsresistant to a compound designated G418; (b) a gene encoding the enzymeα-glucosidase; and (c) a casein promoter element. The transfected cellswere selected for transgenic modification by selecting for transgeniccells in cell culture conditions harboring G418. The transgenic cellsare then screened for transgenic modification by utilizing one or morescreening techniques. Examples of these techniques are: (1) polymerasechain reaction, (2) Southern blotting, and (3) FISH-filter procedures.These techniques are well known to a person of ordinary skill in theart. The latter two techniques are utilized to determine the number ofcopies of an inserted gene sequence in embryonic stem cell nuclear DNA.

These screening procedures can be applied to transfected cells at any ofthe steps indicated in FIG. 4. Cloned transgenic animals may be createdfrom transgenic fetuses. Table 4 shows the cloned bovine animalsproduced by the methods described in Examples 1-8.

TABLE 4 Development of Immortalized and Totipotent Cells Bovine AnimalsProduced Age of Fetus Type of Cell Type of Cell Harvested Used Used Dateof Birth Gender Breed (days) (1° NT) (2° NT) Stimulus  2/6/97 M H 60(est) EG Blastomere LIF, FGF  7/7/97 (*) M H 60 (est) EG Blastomere LIF,FGF  7/7/97 (*) M H 60 (est) EG Blastomere LIF, FGF  2/7/98 F H 55 (est)EG Blastomere LIF, FGF, FSK  2/26/98 F H 55 (est) EG Blastomere LIF,FGF, FSK 10/26/98 F H 55 (est) EG Blastomere LIF, FGF, FSK 10/28/98 F H58 EG Blastomere LIF, FGF, FSK 12/2/98 F BS 58 EG n/a LIF, FGF, FSK12/6/98 F H 58 EG n/a Culture 12/22/98 (*) F H 58 EG n/a Culture12/22/98 F H 58 EG n/a Culture 12/22/98 (*) F H 58 EG (t) n/a LIF, FGF12/30/98 F H 58 EG n/a Culture 12/30/98 F H 58 EG n/a Culture 12/31/98 FH 58 EG n/a Culture  1/6/99 F H 58 EG (t) n/a LIF, FGF  1/7/99 F H 58 EGn/a Culture  1/7/99 F H 58 EG n/a Culture  1/15/99 F H 58 EG n/a Culture 1/15/99 F H 58 EG (t) n/a LIF, FGF  1/19/99 F H 58 EG (t) n/a LIF, FGF(*) - Stillborn H - Holstein BS - Brown Swiss Age of fetus harvested -Age (in days) of fetus used as a source of precursor cells in 1° NT(t) - transgenic nuclear donor: a nuclear donor cell transfected with aDNA construct having a human α glucosidase gene EGF - Epidermal growthfactor LIF - Leukemia inhibitor factor FSK - Forskolin

While the invention has been described and exemplified in sufficientdetail for those skilled in this art to make and use it, variousalternatives, modifications, and improvements should be apparent withoutdeparting from the spirit and scope of the invention.

One skilled in the art readily appreciates that the present invention iswell adapted to carry out the objects and obtain the ends and advantagesmentioned, as well as those inherent therein. The cell lines, embryos,animals, and processes and methods for producing them are representativeof preferred embodiments, are exemplary, and are not intended aslimitations on the scope of the invention. Modifications therein andother uses will occur to those skilled in the art. These modificationsare encompassed within the spirit of the invention and are defined bythe scope of the claims.

It will be readily apparent to a person skilled in the art that varyingsubstitutions and modifications may be made to the invention disclosedherein without departing from the scope and spirit of the invention.

All patents and publications mentioned in the specification areindicative of the levels of those of ordinary skill in the art to whichthe invention pertains. All patents and publications are hereinincorporated by reference to the same extent as if each individualpublication was specifically and individually indicated to beincorporated by reference.

The invention illustratively described herein suitably may be practicedin the absence of any element or elements, limitation or limitationswhich is not specifically disclosed herein. Thus, for example, in eachinstance herein any of the terms “comprising”, “consisting essentiallyof” and “consisting of” may be replaced with either of the other twoterms. The terms and expressions which have been employed are used asterms of description and not of limitation, and there is no intentionthat in the use of such terms and expressions of excluding anyequivalents of the features shown and described or portions thereof, butit is recognized that various modifications are possible within thescope of the invention claimed. Thus, it should be understood thatalthough the present invention has been specifically disclosed bypreferred embodiments and optional features, modification and variationof the concepts herein disclosed may be resorted to by those skilled inthe art, and that such modifications and variations are considered to bewithin the scope of this invention as defined by the appended claims.

In addition, where features or aspects of the invention are described interms of Markush groups, those skilled in the art will recognize thatthe invention is also thereby described in terms of any individualmember or subgroup of members of the Markush group. For example, if X isdescribed as selected from the group consisting of bromine, chlorine,and iodine, claims for X being bromine and claims for X being bromineand chlorine are fully described.

Other embodiments are set forth within the following claims.

What is claimed is:
 1. A method for preparing an ungulate animal, saidmethod comprising: a) forming a first embryo by nuclear transfer of anungulate cell obtained from a cell culture of a non-embryonic cell, or anucleus thereof, into a first enucleated oocyte obtained from the samespecies as the ungulate cell; b) forming a second embryo by nucleartransfer of a cell obtained from said first embryo, or a nucleusthereof, into a second enucleated oocyte obtained from the same speciesas the cell in step (a) wherein said cell obtained from said firstembryo is not expanded in culture; and c) transferring said secondembryo into the uterus of an ungulate of the same species as the cell instep (a) so as to produce a fetus that undergoes full fetal developmentand parturition to generate said ungulate animal.
 2. The method of claim1, wherein said ungulate animal is an individual of a species selectedfrom the group consisting of ovine, caprine, and bovine.
 3. The methodof claim 1, wherein said cell culture is contacted with one or moremedia components selected from the group consisting of cytokine, growthfactor, trophic factor, and neurotrophic factor.
 4. The method of claim1, wherein said cell culture is contacted with one or more natural orrecombinant media components selected from the group consisting of humanLIF, bovine LIF, bFGF, oncostatin M, ciliary neurotrophic factor,cardiotrophin 1, stem cell factor, IL-6, IL-11, IL-12, and forskolin. 5.The method of claim 1, wherein said cell culture is contacted with LIFand FGF.
 6. The method of claim 1, wherein said cell culture iscontacted with LIF, FGF and forskolin.
 7. A method according to claim 1,wherein said cell obtained from said cell culture is a transgenic cell.8. A method according to claim 1, wherein said nuclear transfer in step(a) comprises: (i) fusing said cell obtained from said cell culture, ora nucleus thereof, and said first enucleated oocyte to form a fusedcell; and (ii) activating said fused cell.
 9. A method according toclaim 1, wherein said nuclear transfer in step (b) comprises: (i) fusingsaid cell obtained from said first embryo, or a nucleus thereof, andsaid second enucleated oocyte to form a fused cell; and (ii) activatingsaid fused cell.
 10. A method according to claim 8 or 9, wherein saidfusion of step (i) comprises using an electrical stimulus.
 11. A methodaccording to claim 8 or 9, wherein said activation of step (ii)comprises increasing intracellular levels of divalent cations andreducing phosphorylation of cellular proteins in said fused cell.
 12. Amethod according to claim 11, wherein said activation of step (ii)comprises incubating said fused cell in a medium comprising ionomycinand/or 6-dimethylaminopurine.
 13. A method according to claim 1, whereinsaid cell culture is a fetal cell culture.
 14. A method according toclaim 13, wherein said fetal cell is a somatic cell or a genital ridgecell.
 15. A method according to claim 1, wherein said cell culture isobtained by culture of one or more cells obtained from an ex uteroungulate animal.
 16. A method according to claim 15, wherein said adultcell is selected from the group consisting of an amniotic cell, afibroblast cell, an epithelial cell, and a cumulus cell.
 17. A methodaccording to claim 1, wherein said cell obtained from said cell cultureis not serum starved.
 18. A method according to claim 1, wherein saidfirst embryo is cultured, such that said first embryo comprises two ormore cells.
 19. A method for reconstructing an ungulate embryo, saidmethod comprising: a) forming a first embryo by nuclear transfer of anungulate cell obtained from a cell culture, or a nucleus thereof, into afirst enucleated oocyte obtained from the same species as the cell in;and b) forming a second embryo by nuclear transfer of a cell obtainedfrom said first embryo, or a nucleus thereof, into a second enucleatedoocyte obtained from the same species as the cell in step (a).
 20. Themethod of claim 19, wherein said ungulate embryo is of a speciesselected from the group consisting of ovine, caprine, and bovine. 21.The method of claim 19, wherein said cell culture is contacted with oneor more media components selected from the group consisting of cytokine,growth factor, trophic factor, and neurotrophic factor.
 22. The methodof claim 19, wherein said cell culture is contacted with one or morenatural or recombinant media components selected from the groupconsisting of human LIF, bovine LIF, bFGF, oncostatin M, ciliaryneurotrophic factor, cardiotrophin 1, stem cell factor, IL-6, IL-11,IL-12, and forskolin.
 23. The method of claim 19, wherein said cellculture is contacted with LIF and FGF.
 24. The method of claim 19,wherein said cell culture is contacted with LIF, FGF and forskolin. 25.A method according to claim 19, wherein said cell obtained from saidcell culture is a transgenic cell.
 26. A method according to claim 19,wherein said nuclear transfer in step (a) comprises: (i) fusing saidcell obtained from said cell culture, or a nucleus thereof, and saidfirst enucleated oocyte to form a fused cell; and (ii) activating saidfused cell.
 27. A method according to claim 19, wherein said nucleartransfer in step (b) comprises: (i) fusing said cell obtained from saidfirst embryo, or a nucleus thereof, and said second enucleated oocyte toform a fused cell; and (ii) activating said fused cell.
 28. A methodaccording to claim 26 or 27, wherein said fusion of step (i) comprisesusing an electrical stimulus.
 29. A method according to claim 26 or 27,wherein said activation of step (ii) comprises increasing intracellularlevels of divalent cations and reducing phosphorylation of cellularproteins in said fused cell.
 30. A method according to claim 27, whereinsaid activation of step (ii) comprises incubating said fused cell in amedium comprising ionomycin and/or 6-dimethylaminopurine.
 31. A methodaccording to claim 19, wherein said cell culture is a fetal cellculture.
 32. A method according to claim 31, wherein said fetal cell isa somatic cell or a genital ridge cell.
 33. A method according to claim19, wherein said cell culture is obtained by culture of one or morecells obtained from an ex utero ungulate animal.
 34. A method accordingto claim 33, wherein said adult cell is selected from the groupconsisting of an amniotic cell, a fibroblast cell, an epithelial cell,and a cumulus cell.
 35. A method according to claim 19, wherein saidcell obtained from said cell culture is not serum starved.
 36. A methodaccording to claim 19, wherein said first embryo is cultured, such thatsaid first embryo comprises two or more cells.
 37. A method forpreparing an ungulate animal, said method comprising: a) forming a firstembryo by nuclear transfer of an ungulate cell obtained from a firstcell culture of a non-embryonic cell, or a nucleus thereof, into a firstenucleated oocyte obtained from the same species as the ungulate cell;b) transferring said first embryo into the uterus of an ungulate of thesame species as the cell in step (a) so as to produce a first fetus; c)forming a second cell culture by culturing one or more cells obtainedfrom said first fetus; d) forming a second embryo by nuclear transfer ofa cell obtained from said second cell culture, or a nucleus thereof,into a second enucleated oocyte obtained from the same species as thecell in step (a); and e) transferring said second embryo into the uterusof an ungulate of the same species as the cell in step (a) so as toproduce a second fetus that undergoes full fetal development andparturition to generate said ungulate animal.
 38. The method of claim37, wherein said ungulate animal is an individual of a species selectedfrom the group consisting of ovine, caprine, and bovine.
 39. The methodof claim 37, wherein said first and/or said second cell culture iscontacted with one or more media components selected from the groupconsisting of cytokine, growth factor, trophic factor, and neurotrophicfactor.
 40. The method of claim 37, wherein said first and/or saidsecond cell culture is contacted with one or more natural or recombinantmedia components selected from the group consisting of human LIF, bovineLIF, bFGF, oncostatin M, ciliary neurotrophic factor, cardiotrophin 1,stem cell factor, IL-6, IL-11, IL-12, and forskolin.
 41. The method ofclaim 37, wherein said first and/or said second cell culture iscontacted with LIF and FGF.
 42. The method of claim 37, wherein saidfirst and/or said second cell culture is contacted with LIF, FGF andforskolin.
 43. A method according to claim 37, wherein said cellobtained from said first and/or said second cell culture is a transgeniccell.
 44. A method according to claim 37, wherein said nuclear transferin step (a) comprises: (i) fusing said cell obtained from said firstcell culture, or a nucleus thereof, and said first enucleated oocyte toform a fused cell; and (ii) activating said fused cell.
 45. A methodaccording to claim 37, wherein said nuclear transfer in step (d)comprises: (i) fusing said cell obtained from said second cell culture,or a nucleus thereof, and said second enucleated oocyte to form a fusedcell; and (ii) activating said fused cell.
 46. A method according toclaim 44 or 45, wherein said fusion of step (i) comprises using anelectrical stimulus.
 47. A method according to claim 44 or 45, whereinsaid activation of step (ii) comprises increasing intracellular levelsof divalent cations and reducing phosphorylation of cellular proteins insaid fused cell.
 48. A method according to claim 47, wherein saidactivation of step (ii) comprises incubating said fused cell in a mediumcomprising ionomycin and/or 6-dimethylaminopurine.
 49. A methodaccording to claim 37, wherein said first cell culture is a fetal cellculture.
 50. A method according to claim 49, wherein said fetal cell isa somatic cell or a genital ridge cell.
 51. A method according to claim37, wherein said first cell culture is an adult cell culture.
 52. Amethod according to claim 51, wherein said adult cell is selected fromthe group consisting of an amniotic cell, a fibroblast cell, anepithelial cell, and a cumulus cell.
 53. A method according to claim 37,wherein said cell obtained from said first and/or said second cellculture is not serum starved.
 54. A method according to claim 37,wherein said first embryo is cultured, such that said first embryocomprises two or more cells.
 55. A method according to claim 37, whereinsaid one or more cells obtained from said first fetus are somatic cellsor genital ridge cells.
 56. A method according to claim 37, wherein saidcell obtained from said second cell culture is not serum starved.
 57. Amethod according to claim 37, wherein said second embryo is cultured,such that said second embryo comprises two or more cells.